Intermediates for the synthesis of bile acid derivatives, in particular of obeticholic acid

ABSTRACT

The present invention relates to compounds which are intermediates in the synthesis of bile acid derivatives with pharmacological activity. The invention relates to compounds of general formula (I): 
                         
wherein:
 
 , R 1 , R 2 , R 3 , R 4 , R 5 , R 6  and Y are as defined herein. The compounds are intermediates in the synthesis of synthetic bile acids which are useful in the treatment of conditions such as liver disease. In addition, the invention relates to a method of synthesizing these intermediates and a method of preparing obeticholic acid and obeticholic acid analogues from the compounds of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of International Application No.PCT/GB2017/051385, filed May 18, 2017, which claims the benefit of GBPatent Application No. 1608777.7 filed May 18, 2016, the entire contentsof which are hereby incorporated by reference herein in theirentireties.

The present invention relates to compounds which are intermediates inthe synthesis of bile acid derivatives with pharmacological activity. Inparticular, the invention relates to intermediates in the synthesis ofobeticholic acid and its analogues. In addition, the invention relatesto a method of synthesizing these intermediates and a method ofpreparing obeticholic acid and obeticholic acid analogues from thecompounds of the invention.

Bile acids are steroid acids which are found in the bile of mammals andinclude compounds such as cholic acid, chenodeoxycholic acid,lithocholic acid and deoxycholic acid, all of which are found in humans.Many bile acids are natural ligands of the farnesoid X receptor (FXR)which is expressed in the liver and intestine of mammals, includinghumans.

Bile acids are derivatives of steroids and are numbered in the same way.The following shows the general numbering system for steroids and thenumbering of the carbon atoms in chenodeoxycholic acid.

Agonists of FXR have been found to be of use in the treatment ofcholestatic liver disorders including primary biliary cirrhosis andnon-alcoholic steatohepatitis (see review by Jonker et al, in Journal ofSteroid Biochemistry & Molecular Biology, 2012, 130, 147-158).

Ursodeoxycholic acid (UDCA), a bile acid originally isolated from thegall bladder of bears, is currently used in the treatment of cholestaticliver disorders, although it appears to be inactive at the FXR.

As well as their action at the FXR, bile acids and their derivatives arealso modulators of the G protein-coupled receptor TGR5. This is a memberof the rhodopsin-like superfamily of G-protein coupled receptors and hasan important role in the bile acid signalling network, which complementsthe role of the FXR.

Because of the importance of FXR and TGR5 agonists in the treatment ofcholestatic liver disorders, efforts have been made to develop newcompounds which have agonist activity at these receptors. Oneparticularly active compound is obeticholic acid, which is a potentagonist of both FXR and TGR5. Obeticholic acid is described in WO02/072598 and EP1568706, both of which describe a process for thepreparation of obeticholic acid from 7-keto lithocholic acid, which isderived from cholic acid. Further processes for the production ofobeticholic acid and its derivatives are described in WO 2006/122977, US2009/0062256 and WO 2013/192097 and all of these processes also startfrom 7-keto lithocholic acid.

It is clear from the number of patent publications directed to processesfor the production of obeticholic acid that it is by no means simple tosynthesise this compound and indeed the process which is currently usedstarts from cholic acid, has 12 steps and a low overall yield.

In addition to the inefficiency and high cost of this process, there arealso problems with the cost and availability of the starting materials.Cholic acid, the current starting material for the production ofobeticholic acid, is a natural bile acid which is usually obtained fromthe slaughter of cows and other animals. This means that theavailability of cholic acid and other bile acids is limited by thenumber of cattle available for slaughter. Since the incidence ofcholestatic liver disease is increasing worldwide, the demand forsynthetic bile acids such as obeticholic acid is also likely to increaseand it is doubtful whether the supply of naturally derived bile acidswill continue to be sufficient to meet demand.

Furthermore, the use of a starting material derived from animals meansthat there is the possibility of the contamination of the material withinfectious agents such as viruses or prions, which can not only behazardous to workers but could potentially contaminate the end productsif steps are not taken to prevent this.

Although some patients with cholestatic liver disease can be treatedwith ursodeoxycholic acid, this is also a natural bile acid and facesthe same problems of limited availability and high cost.

In an attempt to solve the problems associated with the use of bileacids as starting materials, the present inventors have devised aprocess for the synthesis of synthetic bile acid derivatives, such asobeticholic acid, which uses plant sterols as starting materials.

The inventors have developed a process for the production of syntheticbile acids which proceeds via novel intermediates and which provides thefinal product in significantly higher yield than current processes. Theprocess is flexible and can use a variety of different startingmaterials including animal, fungal and plant sterols.

Suitable animal sterols which can be used as starting materials includedeoxycholic acid, cholic acid, while fungal sterols include ergosterol.

Plant sterols are widely available at significantly lower cost than bileacids and, indeed, are often waste products of other processes. Suitableplant sterol and plant sterol derivatives which can be used as startingmaterials include 3-keto-bis-norcholenol (also known as20-hydroxymethylpregn-4-en-3-one), androstenedione, androstadienedione,dehydroepiandrosterone, stigmasterol, brassicasterol, campesterol andR-sitosterol.

Our patent applications Nos. PCT/GB2015/053516 (WO2016/079517),PCT/GB2015/053517 (WO2016/079518), PCT/GB2015/053518 (WO2016/079519) andPCT/GB2015/053519 (WO2016/079520) relate to intermediates in the processas well as to processes for preparing the intermediates and processesfor converting them to the desired products. The present applicationrelates to further compounds which are analogues of the compoundsdescribed in WO2016/079517, WO2016/079518, WO2016/079519 andWO2016/079520.

In the present invention there is provided a compound of general formula(I):

wherein:

is a carbon-carbon single or double bond;

R¹ is C₁₋₄ alkyl optionally substituted with one or more substituentsselected from halo, OR^(7a) and NR^(7a)R^(7b);

-   -   where each of R^(7a) and R^(7b) is independently selected from H        and C₁₋₄ alkyl; or R¹ and R² together form an epoxide group;

R² is ═O or OH or a protected OH or R² and R¹ together form an epoxidegroup;

R³ is H, halo or OH or a protected OH;

when

is a carbon-carbon double bond, Y is a bond or an alkylene, alkenyleneor alkynylene linker group having from 1 to 20 carbon atoms andoptionally substituted with one or more groups R¹³;

when

is a carbon-carbon single bond, Y is a bond or an alkylene linker grouphaving from 1 to 20 carbon atoms and optionally substituted with one ormore groups R¹³;

-   -   each R¹³ is independently halo, OR⁸ or NR⁸R⁹;        -   where each of R⁸ and R⁹ is independently selected from H and            C₁₋₄ alkyl;

R⁴ is halo, CN, C(O)R¹⁰, CH(OR¹⁰)(OR¹¹), CH(R¹⁰)(OR¹¹), CH(SR¹⁰)(SR¹¹),NR¹⁰R¹¹, BR¹⁰R¹¹, C(O)CH₂N₂, —CH═CH₂, —C≡CH, CH[C(O)OR¹⁰]₂,CH(BR¹⁰R¹¹)₂, azide or a carboxylic acid mimetic group;

-   -   where each R¹⁰ and R¹¹ is independently:    -   a. hydrogen or    -   b. C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl or C₂₋₂₀ alkynyl, any of which is        optionally substituted with one or more substituents selected        from halo, NO₂, CN, OR¹⁹, SR¹⁹, C(O)OR¹⁹, C(O)N(R¹⁹)₂, SO₂R¹⁹,        SO₃R¹⁹, OSO₃R¹⁹, N(R¹⁹)₂ and a 6- to 14-membered aryl or 5 to        14-membered heteroaryl group, either of which is optionally        substituted with one or more substituents selected from C₁₋₆        alkyl, C₁₋₆ haloalkyl, halo, NO₂, CN, OR¹⁹, SR¹⁹, C(O)OR¹⁹,        C(O)N(R¹⁹)₂, SO₂R¹⁹, SO₃R¹⁹, OSO₃R¹⁹ and N(R¹⁹)₂; or    -   c. a 6- to 14-membered aryl or 5 to 14-membered heteroaryl group        either of which is optionally substituted with one or more        substituents selected from C₁₋₆ alkyl, C₁₋₆ haloalkyl, halo,        NO₂, CN, OR¹⁹, SR¹⁹, C(O)OR¹⁹, C(O)N(R¹⁹)₂, SO₂R¹⁹, SO₃R¹⁹,        OSO₃R¹⁹ and N(R¹⁹)₂; or    -   d. a polyethylene glycol residue; or    -   e. when R⁴ is CH(OR¹⁰)(OR¹¹), CH(R¹⁰)(OR¹¹), CH(SR¹⁰)(SR¹¹),        NR¹⁰R¹¹, BR¹⁰R¹¹, CH[C(O)OR¹⁰]₂ or CH(BR¹⁰R¹¹)₂ an R¹⁰ and an        R¹¹ group, together with the atom or atoms to which they are        attached, may combine to form a 3 to 10-membered heterocyclic        ring;        -   each R¹⁹ is independently selected from H, C₁₋₆ alkyl, C₁₋₆            haloalkyl and a 6- to 14-membered aryl or 5 to 14-membered            heteroaryl group either of which is optionally substituted            with one or more substituents selected from halo, C₁₋₆ alkyl            and C₁₋₆ haloalkyl; or

Y and R⁴ together form a ═CH₂ group;

R⁵ is H or OH or a protected OH group;

R⁶ is ═O;

or a salt or an isotopic variant thereof.

Compounds of general formula (I) are intermediates in the synthesis ofpharmaceutically active compounds such as obeticholic acid and itsderivatives.

In the present specification, except where the context requiresotherwise due to express language or necessary implication, the word“comprises”, or variations such as “comprises” or “comprising” is usedin an inclusive sense i.e. to specify the presence of the statedfeatures but not to preclude the presence or addition of furtherfeatures in various embodiments of the invention.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

In the present application the term “C₁₋₂₀” alkyl refers to a straightor branched fully saturated hydrocarbon group having from 1 to 20 carbonatoms. The term encompasses methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, s-butyl and t-butyl. Other alkyl groups, for example C₁₋₁₂alkyl, C₁₋₁₀ alkyl, C₁₋₆ alkyl or C₁₋₃ alkyl are as defined above butcontain different numbers of carbon atoms.

The terms “heterocyclic” and “heterocyclyl” refer to a non-aromaticcyclic group having 3 to 10 ring atoms and at least one heteroatomselected from N, O, S and B and optionally substituted with one or more═O moieties. Examples of heterocyclic groups include pyrrolidine,piperidine, morpholine, piperazine, tetrahydrofuran, dioxolane (e.g.1,3-dioxolane), dioxane (e.g. 1,3-dioxane) and cyclic thioethers. Theterm also includes bicyclic and bridged groups such as9-borabicyclo(3.3.1)nonane (9-BBN).

The term “halogen” refers to fluorine, chlorine, bromine or iodine andthe term “halo” to fluoro, chloro, bromo or iodo groups.

The term “C₁₋₆ haloalkyl” refers to a straight or branched alkyl groupas defined above having from 1 to 6 carbon atoms and substituted withone or more halo atoms, up to perhalo substitution. Examples includetrifluoromethyl, chloroethyl and 1,1-difluoroethyl.

The term “C₂₋₂₀ alkenyl” refers to a straight or branched hydrocarbongroup having from 2 to 20 carbon atoms and at least one carbon-carbondouble bond. Examples include ethenyl, prop-1-enyl, hex-2-enyl etc.Other alkenyl groups, for example C₂₋₁₂ alkenyl, C₂₋₁₀ alkenyl, C₂₋₈alkenyl, C₂₋₆ alkenyl, C₂₋₅ alkenyl, C₂₋₄ alkenyl or C₂₋₃ alkenyl are asdefined above but contain different numbers of carbon atoms.

The term “C₂₋₂₀ alkynyl” refers to a straight or branched hydrocarbongroup having from 2 to 20 carbon atoms and at least one carbon-carbontriple bond. Examples include ethynyl, prop-1-ynyl, hex-2-ynyl etc.Other alkynyl groups, for example C₂₋₁₂ alkynyl, C₂₋₁₀ alkynyl, C₂₋₈alkynyl, C₂₋₆ alkynyl, C₂₋₅ alkynyl, C₂₋₄ alkynyl or C₂₋₃ alkynyl are asdefined above but contain different numbers of carbon atoms.

The term “alkylene” refers to a straight or branched fully saturatedhydrocarbon chain. Suitably alkylene is C₁₋₂₀ alkylene, C₁₋₁₂ alkylene,C₁₋₁₀ alkylene, C₁₋₈ alkylene, C₁₋₆ alkylene, C₁₋₅ alkylene, C₁₋₄alkylene, C₁₋₃ alkylene, or C₁₋₂ alkylene. Examples of alkylene groupsinclude —CH₂—, —CH₂CH₂—, —CH(CH₃)—CH₂—, —CH₂CH(CH₃)— —CH₂CH₂CH₂—,—CH₂CH(CH₂CH₃)— and —CH₂CH(CH₂CH₃)CH₂—.

The term “alkenylene” refers to a straight or branched hydrocarbon chaincontaining at least one carbon-carbon double bond. Suitably alkenyleneis C₂₋₂₀ alkenylene, C₂₋₁₂ alkenylene, C₂₋₁₀ alkenylene, C₂₋₈alkenylene, C₂₋₆ alkenylene, C₂₋₅ alkenylene, C₂₋₄ alkenylene, or C₂₋₃alkenylene. Examples of alkenylene groups include —CH═CH—, —CH═C(CH₃)—,—CH₂CH═CH—, —CH═CHCH₂—, —CH₂CH₂CH═CH—, —CH₂CH═C(CH₃)— and—CH₂CH═C(CH₂CH₃)—.

The term “alkynylene” refers to a straight or branched hydrocarbon chaincontaining at least one carbon-carbon triple bond. Suitably alkynyleneis C₂₋₂₀ alkynylene, C₂₋₁₂ alkynylene, C₂₋₁₀ alkynylene, C₂₋₈alkynylene, C₂₋₆ alkynylene, C₂₋₅ alkynylene, C₂₋₄ alkynylene, or C₂₋₃alkynylene. Examples of alkynylene groups include —C≡C—, —CH₂C≡C—,—C≡C—CH₂—, —CH₂CH₂C≡C—, —CH₂C≡CCH₂— and —CH₂C—C≡CH₂CH₂—.

The terms “aryl” and “aromatic” refer to a cyclic group with aromaticcharacter having from 6 to 14 ring carbon atoms (unless otherwisespecified) and containing up to three rings. Where an aryl groupcontains more than one ring, not all rings must be aromatic incharacter. Examples include phenyl, naphthyl and anthracenyl as well aspartially saturated systems such as tetrahydronaphthyl, indanyl andindenyl.

The terms “heteroaryl” and “heteroaromatic” refer to a cyclic group witharomatic character having from 5 to 14 ring atoms (unless otherwisespecified), at least one of which is a heteroatom selected from N, O andS, and containing up to three rings. Where a heteroaryl group containsmore than one ring, not all rings must be aromatic in character.Examples of heteroaryl groups include pyridine, pyrimidine, indole,benzofuran, benzimidazole and indolene.

The term “isotopic variant” refers to isotopically-labelled compoundswhich are identical to those recited in formula (I) but for the factthat one or more atoms are replaced by an atom having an atomic mass ormass number different from the atomic mass or mass number most commonlyfound in nature, or in which the proportion of an atom having an atomicmass or mass number found less commonly in nature has been increased(the latter concept being referred to as “isotopic enrichment”).Examples of isotopes that can be incorporated into compounds of theinvention include isotopes of hydrogen, carbon, nitrogen, oxygen,fluorine, iodine and chlorine such as ²H (deuterium), ³H, ¹¹C, ¹³C, ¹⁴C,¹⁸F, ¹²³I or ¹²⁵I, which may be naturally occurring or non-naturallyoccurring isotopes.

Polyethylene glycol (PEG) is a polyether compound, which in linear formhas general formula H—[O—CH₂—CH₂]_(n)—OH. A polyethylene glycol residueis a PEG in which the terminal H is replaced by a bond linking it to theremainder of the molecule. Branched versions, including hyperbranchedand dendritic versions are also contemplated and are generally known inthe art. Typically, a branched polymer has a central branch core moietyand a plurality of linear polymer chains linked to the central branchcore. PEG is commonly used in branched forms that can be prepared byaddition of ethylene oxide to various polyols, such as glycerol,glycerol oligomers, pentaerythritol and sorbitol. The central branchmoiety can also be derived from several amino acids, such as lysine. Thebranched poly (ethylene glycol) can be represented in general form asR(-PEG-OH)_(m) in which R is derived from a core moiety, such asglycerol, glycerol oligomers, or pentaerythritol, and m represents thenumber of arms. Multi-armed PEG molecules, such as those described inU.S. Pat. Nos. 5,932,462; 5,643,575; 5,229,490; 4,289,872; US2003/0143596; WO 96/21469; and WO 93/21259 may also be used.

The PEG polymers may have an average molecular weight of, for example,600-2,000,000 Da, 60,000-2,000,000 Da, 40,000-2,000,000 Da,400,000-1,600,000 Da, 800-1,200,000 Da, 600-40,000 Da, 600-20,000 Da,4,000-16,000 Da, or 8,000-12,000 Da.

The term “protected OH” relates to an OH group protected with anysuitable protecting group.

Suitable protecting groups include esters such that, for example when R²and/or R³ and/or R⁵ is a protected OH group, R² and/or R³ and/or R⁵and/or R⁶ may independently be a group OC(O)R¹⁴, where R¹⁴ is a groupR¹⁰ as defined above.

Silyl ethers are also suitable, and in this case, R² and/or R³ and/or R⁵may independently be a group OSi(R¹⁶)₃, where each R¹⁶ is independently:

a. C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl or C₂₋₂₀ alkynyl optionally substitutedwith one or more substituents selected from halo, NO₂, CN, OR¹⁹, SR¹⁹,SO₂R¹⁹, SO₃R¹⁹ or N(R¹⁹)₂, a 6- to 14-membered aryl or 5 to 14-memberedheteroaryl group, either of which is optionally substituted with C₁₋₆alkyl, C₁₋₆ haloalkyl, halo, NO₂, CN, OR¹⁹, SO₂R¹⁹, SO₃R¹⁹ or N(R¹⁹)₂;or

b. a 6- to 14-membered aryl or 5 to 14-membered heteroaryl groupoptionally substituted with one or more substituents selected from C₁₋₆alkyl, C₁₋₆ haloalkyl, halo, NO₂, CN, OR¹⁹, SR¹⁹, SO₂R¹⁹, SO₃R¹⁹ orN(R¹⁹)₂;

each R¹⁹ is independently selected from H, C₁₋₆ alkyl or C₁₋₆ haloalkyl.

Silyl ethers are particularly suitable for the protection of OH at theR² position (J. Med. Chem., 2014, 57, 937-954). In this case, it isparticularly suitable for each R¹⁶ to be independently C₁₋₆ alkyl. Anexample of a silyl protected OH group R² is t-butyldimethylsilyloxy.

Other suitable protecting groups for OH are well known to those of skillin the art (see Wuts, P G M and Greene, T W (2006) “Greene's ProtectiveGroups in Organic Synthesis”, 4^(th) Edition, John Wiley & Sons, Inc.,Hoboken, N.J., USA).

References to a protecting group which is stable in basic conditionsmean that the protecting group cannot be removed by treatment with abase.

Appropriate salts of the compounds of general formula (I) include basicaddition salts such as sodium, potassium, calcium, aluminium, zinc,magnesium and other metal salts as well as choline, diethanolamine,ethanolamine, ethyl diamine, meglumine and other well-known basicaddition salts as summarised in Paulekuhn et al., J. Med. Chem. 2007,50, 6665-6672 and/or known to those skilled in the art.

The term “carboxylic acid mimetic group” relates to known carboxylicacid isosteres including tetrazole, —SO₂—NHR³⁰, C(O)NH—SO₂R³⁰,NHC(O)NH—SO₂R³⁰;

wherein R³⁰ is H, C₁₋₆ alkyl C₃₋₇ cycloalkyl or aryl (e.g. phenyl)optionally substituted, for example with C₁₋₄ alkyl, halo, OH, O(C₁₋₄alkyl), SO₂(C₁₋₄ alkyl), SO₂-phenyl or SO₂-tolyl. Tetrazole groupsinclude tetrazole-5-yl and tetrazole-1-yl and are optionallysubstituted, for example with C₁₋₄ alkyl, halo, OH, O(C₁₋₄ alkyl),SO₂(C₁₋₄ alkyl), SO₂-phenyl or SO₂-tolyl.

Such carboxylic acid mimetic groups are well known in the art and arediscussed, for example in “On Medicinal Chemistry”; M Stocks, L Alcaraz,E Griffen; Pub: Sci-ink Ltd (April 2007).

Particularly suitable carboxylic acid mimetic groups include tetrazole,C(O)NH—SO₂R³⁰ and NHC(O)NH—SO₂R³⁰, with tetrazole being particularlysuitable.

In some cases, the compound of general formula (I) is a compound ofgeneral formula (IA):

wherein R³, Y, R⁴ and R⁵ are as defined for general formula (I).

In other cases, the compound of general formula (I) is a compound ofgeneral formula (IB):

wherein R¹, R³, Y, R⁴ and R⁵ are as defined for general formula (I).

Alternatively, the compound of general formula (I) may be a compound ofgeneral formula (IC):

wherein R¹, R³, Y, R⁴ and R⁵ are as defined for general formula (I).

The compound of general formula (I) may be a compound of general formula(ID):

wherein R¹, R³, Y, R⁴ and R⁵ are as defined for general formula (I).

The compound of general formula (I) may also be a compound of generalformula (IE):

wherein R¹, R³, Y, R⁴ and R⁵ are as defined for general formula (I).

Compounds of general formula (I) may be converted to compounds ofgeneral formula (IF):

wherein R¹, R³, Y, R⁴ and R⁵ are as defined for general formula (I).

Compounds of general formula (IF) are analogues of obeticholic acid inwhich the side chain has a substituent R⁴ as defined above.

In some suitable compounds of general formulae (I), (IB), (IC), (ID) and(IE):

R¹ is C₁₋₄ alkyl optionally substituted with one or more substituentsselected from halo, OR^(7a) or NR^(7a)R^(7b);

-   -   where each of R^(7a) and R^(7b) is independently selected from H        or C₁₋₄ alkyl.

In more suitable compounds of general formulae (I), (IB), (IC), (ID) and(IE), R¹ may be C₁₋₄ alkyl optionally substituted with one or moresubstituents selected from halo, OR^(7a) or NR^(7a)R^(7b), where R^(7a)and R^(7b) are each independently H, methyl or ethyl, especially H ormethyl.

More suitably, R¹ is unsubstituted C₁₋₄ alkyl.

In particularly suitable compounds, R¹ is ethyl.

In some compounds of general formulae (I), (IA), (IB), (IC), (ID) or(IE), Y is a bond.

In some compounds of general formulae (I), (IA), (IB), (IC), (ID) or(IE), Y and R⁴ together form a ═CH₂ group.

In other compounds of general formula (I), particularly compounds offormula (IA) and (IB), Y is an alkylene or alkenylene linker grouphaving from 1 to 15 carbon atoms, more suitably 1 to 12, 1 to 10 or 1 to8 carbon atoms and optionally substituted with one or more groups R¹³ asdefined above. Typically, each R¹³ is independently halo, OR⁸ or NR⁸R⁹;where each of R⁸ and R⁹ is independently selected from H, methyl orethyl, especially H or methyl.

In some more suitable compounds of general formula (I), particularlycompounds of formulae (IA) and (IB), Y is a bond or an unsubstitutedalkylene or alkenylene linker having from 1 to 15 carbon atoms, moresuitably 1 to 12, 1 to 10 or 1 to 8 carbon atoms.

In other more suitable compounds of general formula (I), particularlycompounds of formulae (IA) and (IB), Y is a bond, an unsubstituted C₁₋₃alkylene group, a C₁₋₃ alkylene group substituted with OH, or a C₁₋₃alkenylene group. For example, Y may be a bond, —CH₂—, —CH₂—CH₂—,—CH(OH)—CH₂—, —CH═CH— or —CH═C(CH₃)—, especially —CH₂—, —CH₂—CH₂—,—CH═CH— or —CH═C(CH₃)—.

In other suitable compounds of general formula (I), particularlycompounds of general formulae (IC), (ID) and (IE), Y is an alkylenelinker group having from 1 to 15 carbon atoms, more suitably 1 to 12, 1to 10 or 1 to 8 carbon atoms and optionally substituted with one or moregroups R¹³ as defined above. Typically, each R¹³ is independently halo,OR⁸ or NR⁸R⁹; where each of R⁸ and R⁹ is independently selected from H,methyl or ethyl, especially H or methyl.

In some more suitable compounds of general formula (I), particularlycompounds of general formulae (IC), (ID) and (IE), Y is a bond or anunsubstituted alkylene linker having from 1 to 15 carbon atoms, moresuitably 1 to 12, 1 to 10 or 1 to 8 carbon atoms.

In other more suitable compounds of general formula (I), particularlycompounds of formulae (IC), (ID) and (IE), Y is a bond or an alkylenegroup having 1 to 3 carbon atoms and is optionally substituted with oneor two R¹³ groups, wherein R¹³ is suitably OH, for example Y is a bond,—CH₂—, —CH₂—CH₂— or —CH(OH)—CH₂—, especially —CH₂—, or —CH₂—CH₂—.

In some suitable compounds of general formula (I), Y is an alkylenelinker having from 1 to 15 carbon atoms, more suitably 1 to 12, 1 to 10or 1 to 8 carbon atoms and substituted with an OH group. In this case,the OH group may be separated from the R⁴ moiety by a single CH₂ groupsuch that the linker Y is a group Y⁴—CH(OH)—CH₂—, where Y⁴ is as definedfor Y, but is shorter by two carbon atoms. For example, Y may be—CH(OH)—CH₂—.

This Y linker is particularly suitable when R⁴ is CN or R⁴ isCH(OR¹⁰)(OR¹¹) wherein R¹⁰ and R¹¹ are as defined above, butparticularly wherein the OR¹⁰ and OR¹¹ groups together with the carbonatom to which they are attached form a cyclic acetal group, e.g. a1,3-dioxane or 1,3-dioxolane ring.

In some suitable compounds of general formula (I), R³ is H.

In other suitable compounds of general formula (I), R³ is OH.

In still other suitable compounds of general formula (I), R³ is aprotected OH group. When R³ is a protected OH group, it may be a groupwhich is not stable in a basic environment such that treatment with abase converts the protected OH group to OH. Examples of such groups arewell known in the art and include a group OC(O)R¹⁴ as defined above inwhich R¹⁴ is a group R¹⁰ as defined above for general formula (I).

In the compounds of general formula (I), when R³ is other than hydrogen,it is suitably in the “up” position, i.e. in the beta configuration.

Particularly suitable R¹⁴ groups are as defined below for R¹⁰.

Alternatively, R³ may be a protected OH group which is stable in a basicenvironment. Examples of such groups include OSi(R¹⁶)₃, where each R¹⁶is independently:

-   -   a. C₁₋₂₀ alkyl, C₂₋₂₀ alkenyl or C₂₋₂₀ alkynyl optionally        substituted with one or more substituents selected from halo,        NO₂, CN, OR¹⁹, SR¹⁹, C(O)OR¹⁹, C(O)N(R¹⁹)₂, SO₂R¹⁹, SO₃R¹⁹ or        N(R¹⁹)₂, or a 6- to 14-membered aryl or 5 to 14-membered        heteroaryl group, either of which is optionally substituted with        C₁₋₆ alkyl, C₁₋₆ haloalkyl, halo, NO₂, CN, OR¹⁹, SR¹⁹, C(O)OR¹⁹,        C(O)N(R¹⁹)₂, SO₂R¹⁹, SO₃R¹⁹ or N(R¹⁹)₂; or    -   b. a 6- to 14-membered aryl or 5 to 14-membered heteroaryl group        either of which is optionally substituted with one or more        substituents selected from C₁₋₆ alkyl, C₁₋₆ haloalkyl, halo,        NO₂, CN, OR¹⁹, SR¹⁹, C(O)OR¹⁹, C(O)N(R¹⁹)₂, SO₂R¹⁹, SO₃R¹⁹ or        N(R¹⁹)₂;        -   each R¹⁹ is independently selected from H, C₁₋₆ alkyl or            C₁₋₆ haloalkyl.

Suitably each R¹⁶ is independently selected from:

a. C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl or C₂₋₁₀ alkynyl optionally substitutedwith one or more substituents as described above; or

b. a 6- to 10-membered aryl or 5 to 10-membered heteroaryl groupoptionally substituted with one or more substituents as described above.

More suitably, each R¹⁶ is independently selected from:

-   -   a. C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl or C₂₋₁₀ alkynyl optionally        substituted with one or more substituents as described above; or

b. a 6- to 10-membered aryl group optionally substituted with one ormore substituents as described above.

Still more suitably, each R¹⁶ is independently selected from C₁₋₁₀ alkylor phenyl, either of which is optionally substituted as described above.Examples of OSi(R¹⁶)₃ include trimethylsilyl (TMS), triethylsilyl (TES),triphenylsilyl (TPS), tri-isopropylsilyl (TIPS), dimethylhexylsilyl(TDS), tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBDMSor TBS), di-tert-butylmethylsilyl (DTBMS), diethylisopropylsilyl (DEIPS)and dimethylisopropylsilyl (DMIPS), in particular TMS, TES, TIPS, TBDMSand TBDPS.

In the compounds of general formulae (I), (IA), (IB), (IC), (ID) and(IE), R⁴ is halo, CN, C(O)R¹⁰, CH(OR¹⁰)(OR¹¹), CH(R¹⁰)(OR¹¹),CH(SR¹⁰)(SR¹¹), NR¹⁰R¹¹, BR¹⁰R¹¹, C(O)CH₂N₂, —CH═CH₂, —C≡CH,CH[C(O)OR¹⁰]₂ or CH(BR¹⁰R¹¹)₂, azide or a carboxylic acid mimetic groupsuch as tetrazole.

When present in the R⁴ moiety, suitably, each R¹⁰ and R¹¹ isindependently:

a. hydrogen or

b. C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl or C₂₋₁₀ alkynyl, any of which isoptionally substituted with one or more substituents as described above;or

c. a 6- to 10-membered aryl or 5 to 10-membered heteroaryl group eitherof which is optionally substituted with one or more substituents asdescribed above; or

d. a polyethylene glycol residue; or

e. when R⁴ is CH(OR¹⁰)(OR¹¹), CH(R¹⁰)(OR¹¹), CH(SR¹⁰)(SR¹¹), NR¹⁰R¹¹,BR¹⁰R¹¹, CH[C(O)OR¹⁰]₂ or CH(BR¹⁰R¹¹)₂ an R¹⁰ and an R¹¹ group, togetherwith the atom or atoms to which they are attached, may combine to form a3- to 10-membered heterocyclic ring.

More suitably, each R¹⁰ and R¹¹ is independently

a. hydrogen or

b. C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl or, C₂₋₁₀ alkynyl optionally substitutedwith one or more substituents as described above or

c. a 6- to 10-membered aryl group optionally substituted with one ormore substituents as described above; or

e. when R⁴ is NR¹⁰R¹¹, an R¹⁰ and an R¹¹ group, together with thenitrogen to which they are attached, combine to form a pyrrolidine orpiperidine ring or when R⁴ is CH(OR¹⁰)(OR¹¹), the OR¹⁰ and OR¹¹ group,together with the carbon atom to which they are attached, combine toform a cyclic acetal, particularly a 1,3-dioxane or 1,3-dioxolane ring;or when R⁴ is BR¹⁰R¹¹, the R¹⁰ and R¹¹ groups, together with the boronatom to which they are attached combine to form a bridgedboron-containing ring such as 9-BBN.

Additionally, when R⁴ is NR¹⁰R¹¹, R¹⁰ may be H or C₁₋₄ alkyl and R¹¹ maybe a 5-10 membered heteroaryl group such as tetrazole.

Suitable substituents for alkyl, alkenyl and alkynyl R¹⁰ and R¹¹ groupsand alkyl, alkenyl and alkynyl R¹⁶ groups include halo, NO₂, CN, OR¹⁹,SR¹⁹, C(O)OR¹⁹, SO₂R¹⁹, SO₃R¹⁹, OSO₃R¹⁹, N(R¹⁹)₂ and a 6- to 10-memberedaryl or 5 to 14-membered heteroaryl group, either of which is optionallysubstituted with C₁₋₆ alkyl, C₁₋₆ haloalkyl, halo, NO₂, ON, OR¹⁹,SO₂R¹⁹, SO₃R¹⁹ or N(R¹⁹)₂; where R¹⁹ is as defined above.

More suitable substituents for these R¹⁰, R¹¹ and R¹⁶ groups includehalo, OR¹⁹, C(O)OR¹⁹, N(R¹⁹)₂, SO₃R¹⁹, OSO₃R¹⁹ and a 6- to 10-memberedaryl group optionally substituted as described above, more suitablyoptionally substituted with halo, C₁₋₄ alkyl, C₁₋₄ haloalkyl, —O—C₁₋₄alkyl, —O—C₁₋₄ haloalkyl, C(O)OH, SO₂OH, —NH(C₁₋₄ alkyl) or —N(C₁₋₄alkyl)₂; for example fluoro, chloro, methyl, ethyl, trifluoromethyl,methoxy, ethoxy, trifluoromethoxy, C(O)OH, SO₂OH, amino, methyl amino ordimethylamino.

Suitable substituents for aryl and heteroaryl R¹⁰, R¹¹ and R¹⁶ groupsinclude C₁₋₆ alkyl, C₁₋₆ haloalkyl, halo, NO₂, CN, OR¹⁹, SR¹⁹ orN(R¹⁹)₂.

More suitable substituents for these R¹⁰, R¹¹ and R¹⁶ groups includeC₁₋₄ alkyl, C₁₋₄ haloalkyl, halo, OR¹⁹ or N(R¹⁹)₂; in particular, halo,C₁₋₄ alkyl, C₁₋₄ haloalkyl, —O—C₁₋₄ alkyl, —O—C₁₋₄ haloalkyl, —NH(C₁₋₄alkyl) or —N(C₁₋₄ alkyl)₂.

Specific examples of substituents for aryl and heteroaryl R¹⁰, R¹¹ andR¹⁶ groups include fluoro, chloro, methyl, ethyl, trifluoromethyl,methoxy, ethoxy, trifluoromethoxy, amino, methyl amino anddimethylamino.

As set out above, each R¹⁹ is independently selected from H, C₁₋₆ alkyl,C₁₋₆ haloalkyl, or a 6- to 14-membered aryl or 5 to 14-memberedheteroaryl group either of which is optionally substituted with one ormore halo, C₁₋₆ alkyl or C₁₋₆ haloalkyl substituents.

Suitably, R¹⁹ is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl, or a 6- to 10-memberedaryl or 5 to 10-membered heteroaryl group optionally substituted withone or more halo, C₁₋₄ alkyl or C₁₋₄ haloalkyl substituents.

More suitably, R¹⁹ is H, C₁₋₆ alkyl, C₁₋₆ haloalkyl or phenyl optionallysubstituted with one or more halo, C₁₋₄ alkyl or C₁₋₄ haloalkylsubstituents.

Specific examples of R¹⁹ include H, methyl, ethyl, trifluoromethyl orphenyl optionally substituted with one or more fluoro, chloro, methyl,ethyl or trifluoromethyl groups.

Suitably, R⁴ is halo, CN, C(O)R¹⁰, CH(OR¹⁰)(OR¹¹), NR¹⁰R¹¹, BR¹⁰R¹¹,—CH═CH₂, —C≡CH, CH[C(O)OR¹⁰]₂, azide, a carboxylic acid mimetic group orCH(BR¹⁰R¹¹)₂ or Y and R⁴ together form a ═CH₂ group where R¹⁰ and R¹¹are as described above.

In other suitable compounds, R⁴ is halo, CN, C(O)R¹⁰, CH(OR¹⁰)(OR¹¹),NR¹⁰R¹¹, BR¹⁰R¹¹—CH═CH₂, —C≡CH, CH[C(O)OR¹⁰]₂ or CH(BR¹⁰R¹¹)₂ or Y andR⁴ together form a ═CH₂ group where R¹⁰ and R¹¹ are as described above.

More suitably, R⁴ is halo, CN, C(O)R¹⁰, CH(OR¹⁰)(OR¹¹), CH═CH₂, —C≡CH,CH[C(O)OR¹⁰]₂, BR¹⁰R¹¹, azide or a carboxylic acid mimetic group or Yand R⁴ together form a ═CH₂ group;

where R¹⁰ and R¹¹ are as described above.

When R⁴ is a carboxylic acid mimetic group, it is suitably a tetrazolegroup. Other suitable carboxylic acid mimetic groups are known in theart and include C(O)NH—SO₂R³⁰ and NHC(O)NH—SO₂R³⁰, where R³⁰ is asdefined above.

In other more suitable compounds, R⁴ is halo, CN, C(O)R¹⁰,CH(OR¹⁰)(OR¹¹), CH═CH₂, —C≡CH, CH[C(O)OR¹⁰]₂, BR¹⁰R¹¹ or Y and R⁴together form a ═CH₂ group;

where R¹⁰ and R¹¹ are as described above.

In some particularly suitable compounds, R⁴ is halo, CN, C(O)R¹⁰,CH(OR¹⁰)(OR¹¹), NR¹⁰R¹¹, CH[C(O)OR¹⁰]₂ or azide;

where R¹⁰ and R¹¹ are as described above but are suitably eachindependently H or C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl or C₂₋₁₀ alkynyloptionally substituted as described above or, when R⁴ is NR¹⁰R¹¹, R¹¹may also suitably be a heteroaryl group such as tetrazole; or when R⁴ isCH(OR¹⁰)(OR¹¹), the OR¹⁰ and OR¹¹ groups together with the carbon atomto which they are attached may form a cyclic acetal group, particularlya 1,3-dioxane or 1,3-dioxolane group.

In other particularly suitable compounds, R⁴ is halo, CN, C(O)R¹⁰,CH(OR¹⁰)(OR¹¹) or CH[C(O)OR¹⁰]₂;

where R¹⁰ is H or C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl or C₂₋₁₀ alkynyl optionallysubstituted as described above or when R⁴ is CH(OR¹⁰)(OR¹¹), the OR¹⁰and OR¹¹ groups together with the carbon atom to which they are attachedmay form a cyclic acetal group, particularly a 1,3-dioxane or1,3-dioxolane group.

In still other particularly suitable compounds, R⁴ is a carboxylic acidmimetic group, suitably tetrazole.

In some particularly suitable compounds, R⁴ is halo, CN, C(O)R¹⁰,CH(OR¹⁰)(OR¹¹) or CH[C(O)OR¹⁰]₂; where each R¹⁰ and R¹¹ is independentlyH or C₁₋₄ alkyl or R¹⁰ and R¹¹ together with the carbon and oxygen atomsto which they are attached form a 5- or 6-membered cyclic group.

Examples of R⁴ groups include Cl, Br, CN, C(O)H, CH(OR¹⁰)₂, 1,3-dioxane,1,3-dioxolane and CH[C(O)OR¹⁰]₂; where R¹⁰ is methyl, ethyl, n-propyl,isopropyl, n-butyl, s-butyl, iso-butyl or t-butyl.

Other examples of R⁴ groups include azide and tetrazole.

Still further examples of R⁴ groups include —NH-tetrazole, —C(O)NHSO₂R³⁰and —NHC(O)NHSO₂R³⁰; where R³⁰ is as defined above and tetrazolessubstituted as defined above.

In some suitable compounds of general formulae (I), (IA), (IB), (IC),(ID) and (IE), R⁵ is H.

In other suitable compounds of general formulae (I), (IA), (IB), (IC),(ID) and (IE), R⁵ is OH.

In still other suitable compounds of general formulae (I), (IA), (IB),(IC), (ID) and (IE), R⁵ is a protected OH group.

When R⁵ is a protected OH group, it may be a group which is not stablein a basic environment such that treatment with a base converts theprotected OH group to OH. Examples of such groups are well known in theart and include a group OC(O)R¹⁴ as defined above in which R¹⁴ is agroup R¹⁰ as defined above for general formula (I). Particularlysuitable R¹⁴ groups are as defined for R¹⁰ above.

Alternatively, R⁵ may be a protected OH group which is stable in a basicenvironment. Examples of such groups include OSi(R¹⁶)₃, where each R¹⁶is as defined above.

Particularly suitable R¹⁶ groups are as defined above.

Specific compounds of general formula (I) include the following:

-   (6α, 7α, 20S)-20-(1-bromomethyl)-6,7-epoxy-pregn-4-en-3-one;-   (6α, 7α, 20S)-6,7-epoxy-20-(ethylenedioxymethyl)-pregn-4-en-3-one;-   (6α, 7α, 20S)-6,7-epoxy-20-azidomethyl-pregna-4-en-3-one;-   (6α, 7α)-6,7-epoxy-3-oxo-4-cholen-23-carboxy-24-oic acid dimethyl    ester;-   (6β, 7α)-6-ethyl-7-hydroxy-3-oxo-4-cholen-23-carboxy-24-oic acid    dimethyl ester;-   (5β, 6β, 7α)-6-ethyl-7-hydroxy-3-oxo-cholan-23-carboxy-24-oic acid    dimethyl ester;-   (5β, 6β)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic acid dimethyl    ester;-   (5β, 6α)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic acid dimethyl    ester;-   (5β, 6α)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic acid;-   (6α, 7α)-6,7-epoxy-3-oxo-4-choleno-24-nitrile;-   (6β, 7α)-6-ethyl-7-hydroxy-3-oxo-4-choleno-24-nitrile;-   (5β, 6β, 7α)-6-ethyl-7-hydroxy-3-oxo-cholano-24-nitrile;-   (5β, 6β)-3,7-dioxo-6-ethyl-cholano-24-nitrile;-   (3α, 5β, 6β)-6-ethyl-3-hydroxy-7-oxo-cholano-24-nitrile;-   (6α, 7α, 20R)-20-(1-cyanomethyl)-6,7-epoxy-pregn-4-en-3-one;-   (6β, 7α, 20R)-cyanomethyl-6-ethyl-7-hydroxy-4-pregnen-3-one;-   (5β, 6β, 7α, 20R)-cyanomethyl-6-ethyl-7-hydroxy-pregna-3-one;-   (5β, 6β, 20R)-cyanomethyl-6-ethyl-7-oxo-pregna-3-one;-   (6β, 7α,    20S)-20-(ethylenedioxymethyl)-6-ethyl-7-hydroxy-pregna-4-en-3-one;-   (5β, 6β, 7α,    20S)-20-(ethylenedioxymethyl)-6-ethyl-7-hydroxy-pregna-3-one;-   (5β, 6β, 20S)-20-(ethylenedioxymethyl)-6-ethyl-pregna-3,7-dione;-   (5β, 6α, 20S)-20-(ethylenedioxymethyl)-6-ethyl-pregna-3,7-dione;

or salts thereof.

As discussed in greater detail below, compounds of formula (IF) areanalogues of obeticholic acid and similar compounds of general formula(XXI) and may be used as synthetic precursors of such compounds.

Compounds of general formula (IF) may be prepared from compounds ofgeneral formula (IE) by reduction of a compound of general formula (IE)using a suitable reducing agent and, where R³ and/or R⁵ is a protectedOH, optional removal of the protecting group(s), to give a compound ofgeneral formula (IF) as defined above, wherein removal of the protectinggroup can take place before or after the reduction.

The reducing agent is typically a hydride, such as sodium borohydridewhich may be used in a solvent such as a mixture of tetrahydrofuran andwater. Typically, this reaction is carried out under basic conditions,for example in the presence of a strong base such as sodium or potassiumhydroxide and at a temperature of about 0 to 110° C., more usually 60 to100° C.

Compounds of general formula (IE) may be prepared from compounds ofgeneral formula (ID) as defined above by epimerisation.

The epimerisation reaction suitably comprises treating the compound ofgeneral formula (ID) with a base. The compound of general formula (ID)may be dissolved in an alcoholic solvent, optionally mixed with waterand contacted with a base, for example sodium or potassium hydroxide ora sodium or potassium alkoxide, typically an ethoxide.

If, in the compound of general formula (ID), R³ and/or R⁵ is a protectedOH, for example a group OC(O)R¹⁴, where R¹⁴ is as defined above but isespecially C₁₋₆ alkyl or phenyl, this will be removed during theepimerisation reaction to give a compound of general formula (IE) inwhich R³ and/or R⁵ is OH. Other protected OH groups which are stable inbasic conditions (for example a group OSi(R¹⁶)₃ where each R¹⁶ isindependently as defined above but is especially C₁₋₆ alkyl or phenyl)may be removed subsequently to give a compound of general formula (IE)in which R³ and/or R⁵ is OH.

A compound of general formula (ID) can be prepared by oxidising acompound of general formula (IC) as defined above using any suitablemethod.

One suitable method is a Dess-Martin periodinane(1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3-(1H)-one) oxidation,which may be carried out in a chlorinated solvent such as chloroform ordichloromethane at a temperature of about −5 to 40° C., suitably 0 to30° C., for example 15 to 25° C., suitably at room temperature.

An alternative oxidation method is oxidation using a hypochlorite, forexample sodium hypochlorite, under acidic conditions, for exampleprovided by acetic acid. The reaction may be carried out in an aqueoussolvent and at a temperature of 0 to 15° C., more usually at about 0 to10° C.

Other oxidation methods include a Jones reaction using sodium dichromateor, more usually, chromic trioxide in dilute sulfuric acid. This processis known to be reliable for the clean conversion of bile acid hydroxylgroups to the corresponding keto derivatives (Bortolini et al, J. Org.Chem., 2002, 67, 5802). Alternatively oxidation may be carried out usingTEMPO ((2,2,6,6-Tetramethyl-piperidin-1-yl)oxy) or a derivative thereof.

Compounds of general formula (IC) may be prepared from compounds ofgeneral formula (IB) as defined above by reduction.

The reduction may be hydrogenation, usually catalytic hydrogenation.Suitable catalysts for the catalytic hydrogenation include apalladium/carbon, palladium/calcium carbonate, palladium/aluminiumoxide, platinum/palladium or Raney nickel catalyst. The reaction may becarried out in an organic solvent, which may be an alcoholic solventsuch as methanol, ethanol or isopropanol; ethyl acetate; pyridine;acetic acid; cyclopentyl methyl ether (CPME), acetonitrile (MeCN) orN,N-dimethylformamide (DMF). The organic solvent may optionally be mixedwith a co-solvent such as acetone or water and/or a base such astriethylamine may also be added.

The choice of catalyst and solvent affects the ratio of the requiredproduct of general formula (IC):

to its isomer of general formula (IG):

It also affects the rate of conversion of the reaction intermediate tothe product.

More suitably, a palladium/carbon or palladium/calcium carbonatecatalyst is used. Typically, in the catalyst the palladium is present inan amount of 5-10% by weight with respect to the weight of the matrix(where the matrix is the carbon, calcium carbonate etc).

Particularly suitable solvents and catalysts used for the reactionincluded a mixture of DMF and MeCN with a palladium/calcium carbonatecatalyst and DMF with a palladium/carbon catalyst.

Hydrogenation of a compound of formula (IB) will also reduce any alkenebonds, if present, in the linker Y.

Compounds of general formula (IB) may be prepared from compounds ofgeneral formula (IA) as defined above by selective alkylation with anorganometallic reagent.

Suitable organometallic reagents include Gilman reagents formed byreaction of an alkyl lithium compound of formula (XXX):R¹—Li  (XXX)

wherein R¹ is as defined for general formula (I);

and a copper (I) salt, particularly a copper (I) halide such as copper(I) iodide.

The reaction may be conducted in an organic solvent such astetrahydrofuran, other ethers such as diethylether or a mixture thereof.

Alternatively, the addition can be carried out using Grignard reagentsR¹MgX, where R¹ is as defined for general formula (I) and X is a halide,for example ethylmagnesium bromide and the reaction is suitablyconducted in the presence of a zinc (II) salt such as zinc chloride anda catalytic amount of a copper (I) or copper(II) salt or complex, forexample copper (I) chloride, copper (II) chloride or a copper(I) orcopper (II) acetylacetonate (acac) complex.

The reaction may be carried out in an organic solvent, for example anether such as THF, 2-methyl THF, methyl tert-butyl ether (tBME), diethylether. Surprisingly, the reaction temperature is not particularlysignificant and while in some cases the reaction may be carried out atreduced temperature, for example at about −25 to 0° C., it has also beensuccessfully conducted at higher temperatures of up to about 55° C.

Compounds of general formula (IA) may be prepared from compounds offormula (II):

wherein R³, R⁴, R⁵ and Y are as defined in general formula (I);

by oxidation, for example using methyltrioxorhenium (MTO),monoperoxypthalate (MMPP) or 3-chloroperoxybenzoic acid, (mCPBA).

The reaction using MMPP may be carried out in an organic solvent such asethyl acetate and if mCPBA is used, the reaction may be carried out in asolvent such as dichloromethane, ethyl acetate or toluene. Suitably, thereaction is conducted at or just below the reflux temperature of thesolvent.

Compounds of general formula (II) may be prepared from compounds ofgeneral formula (III):

wherein R³, R⁴, R⁵ and Y are as defined in general formula (I);

by reaction with an oxidizing agent such as chloranil.

The reaction may be carried out under acidic conditions, for example inthe presence of acetic acid, and in an organic solvent such as toluene.

Analogues of compounds of general formulae (IA), (II) and (III) areknown and, for example Uekawa et al in Biosci. Biotechnol. Biochem.,2004, 68, 1332-1337 describe the synthesis of(22E)-3-oxo-4,22-choladien-24-oic acid ethyl ester from stigmasterolfollowed by its conversion to (22E)-3-oxo-4,6,22-cholatrien-24-oic acidethyl ester, which has the formula:

Uekawa et al then go on to describe the conversion of this compound to(6α, 7α, 22E)-6,7-epoxy-3-oxo-4,22-choladien-24-oic acid ethyl ester, ananalogue of a compound of general formula (IA) in which R³ and R⁵ are H,Y is —CH═CH—, and the group in the R⁴ position is C(O)OCH₂CH₃.

Other compounds of general formulae (IA), (II) and (III) may be preparedby analogous methods from phytosterols similar to stigmasterol.

Stigmasterol and other phytosterols are plant sterols and are readilyavailable or may be prepared by known routes.

Compounds of general formula (III) may also be prepared from compoundsof general formula (IV):

wherein R³, R⁴, R⁵ and Y are as defined in general formula (I);

by reaction with lithium bromide and a base such as lithium carbonate.The reaction may be carried out in a solvent such asN,N-dimethylformamide (DMF) and at a temperature of about 120 to 180° C.

Compounds of general formula (IV) may be obtained by bromination of acompound of general formula (V):

wherein R³, R⁴, R⁵ and Y are as defined in general formula (I);

using, for example bromine in acetic acid.

Compounds of general formula (V) may be prepared from compounds ofgeneral formula (VI):

wherein R³, R⁴, R⁵ and Y are as defined in general formula (I);

by oxidation, typically with a chromium-based oxidizing agent or withsodium hypochlorite.

Analogues of compounds of general formula (VI) in which the group at theR⁴ position is C(O)OR¹⁰b, where R^(10b) is C₁₋₆ alkyl or benzyl may beprepared from analogues of compounds of general formula (VI) in whichthe group at the R⁴ position is C(O)OH by esterification, typically byreaction with an appropriate alcohol under acidic conditions. Theanalogue may be converted to a compound of general formula (VI) byconverting the group C(O)OR^(10b) to a group R⁴ as above defined usingone of the methods described below.

Analogues of general formula (VI) in which the group at the R⁴ positionis C(O)OH and R⁵ is H may be prepared from compounds of general formula(VII):

wherein R³ and Y are as defined in general formula (I);

R^(4a) is C(O)OR^(10b), where R^(10b) is C₁₋₆ alkyl or benzyl; and

R¹² is a protecting group;

by reaction with a reducing agent, typically hydrazine under basicconditions and in an alcoholic or glycolic solvent, for examplediethylene glycol. If required, the side chain may be converted to adesired R⁴ group as above defined, as discussed in greater detail below.

Where R¹² is a protecting group which is stable under basic conditions,the reaction may be followed by a reaction to remove the protectinggroup R¹² to leave an OH group.

Protecting groups for OH are discussed above and, for example, R¹² maybe a group C(O)R¹⁴, where R¹⁴ is as defined above, in particular, C₁₋₆alkyl or benzyl. Silyl ethers are also suitable, and in this case, R¹²may be a group Si(R¹⁶)₃, where R¹⁶ is as defined above but is especiallyC₁₋₆ alkyl or phenyl. Other suitable protecting groups for OH are wellknown to those of skill in the art (see Wuts, P G M and Greene, T W(2006) “Greene's Protective Groups in Organic Synthesis”, 4^(th)Edition, John Wiley & Sons, Inc., Hoboken, N.J., USA).

Particularly suitable R¹² groups include groups which are not stable inthe presence of a base since this removes the need for the additionalstep of removing the protecting group. An example of a group R¹² whichis not stable in basic conditions is a group C(O)R¹⁴ where R¹⁴ is asdefined above, and is in particular C₁₋₆ alkyl or phenyl.

Alternatively, the reaction may be carried out in 2 steps such that thecompound of general formula (VII) is reacted with a compound of generalformula (VIII):R²⁰—NH—NH₂  (VIII)

wherein R²⁰ is a leaving group such as toluene sulfonyl or methanesulfonyl;

to give a compound of general formula (IX):

wherein R³ and Y are as defined in general formula (I);

R^(4a) and R¹² are as defined for general formula (VII);

R²⁰ is as defined for general formula (VIII);

followed by reduction with a suitable reducing agent. Examples ofreducing agents which can be used in this reaction include hydrides suchas sodium borohydride, sodium cyanoborohydride, lithium aluminiumhydride etc.

Compounds of general formula (VII) may be prepared from compounds ofgeneral formula (X):

wherein R³ and Y are as defined in general formula (I)

R^(4a) is as defined above for general formula (VII); and

R¹² is as defined above for general formula (VII) and is suitably—C(O)C₁₋₆ alkyl;

by reaction with an oxidizing agent, for example sodium hypochlorite.

The reaction may be carried out under acidic conditions, for example inthe presence of acetic acid, and in an organic solvent such as ethylacetate.

Compounds of general formula (X) may be prepared from compounds ofgeneral formula (XII):

wherein R³ and Y are as defined for general formula (I);

R^(4a) is as defined above for general formula (VII);

by reaction with an agent suitable to introduce the protecting groupR¹². For example, when R¹² is C(O)R¹⁴, the compound of general formula(XII) may be reacted with a carboxylic acid anhydride or an acidchloride in the presence of a weak base such as pyridine, suitablycatalysed by 4-dimethylaminopyridine (DMAP). The reaction may beconducted in a solvent such as ethyl acetate.

Compounds of general formula (XII) may be prepared by the esterificationof compounds of general formula (XIII):

wherein R³ and Y are as defined for general formula (I);

followed by conversion of the ester group to a group R⁴ as abovedefined.

The esterification reaction may be carried out by reacting the acid ofgeneral formula (XIII) with a suitable alcohol under acidic conditions.

Compounds of general formula (XIII) are known. For example, the compoundof general formula (XIII) in which Y is —CH₂CH₂— and R³ is H isdeoxycholic acid, which is readily available from a number of sources.

Other compounds with different values for Y and R³ can be used asalternative starting materials.

Any of compounds (I) to (VII) and (IX) to (XIII) as defined above can beobtained by converting a side chain carboxylic acid, ester, OH orprotected OH group to a group R⁴ as defined above. This conversion maybe carried out by known methods. For example, in a compound of generalformula (XII), the ester may firstly be reduced to give an alcohol ofgeneral formula (XIV):

wherein Y is Y²—CH₂— and Y² is as defined for Y except that it isshorter in length by at least one carbon atom;

and R³ is as defined above for general formula (I).

The reaction may be carried out in two or more steps. In a first step,OH groups in the compound of general formula (XII) may be protected byreaction with a compound of general formula (XV):X¹—Si(R¹⁶)₃  (XV)

wherein R¹⁶ is as defined above and X¹ is a leaving group, for example ahalide such as chloride or a sulfonate leaving group such astrifluoromethanesulfonate (triflate), methanesulfonate (mesylate) ortoluene sulfonate (tosylate);

in the presence of a base such as 2,6-lutidine or triethylamine. In thesecond step, the product of this reaction is reduced, suitably using ahydride such as lithium aluminium hydride or lithium borohydride. Thisreaction is suitably conducted in an organic solvent, for example amixture of methanol and tetrahydrofuran.

Analogues of compounds of general formula (III) in which R⁴ is OH canalso be prepared from plant sterols. For example, Scheme 3 of Example 1illustrates the preparation of a compound of general formula (III) fromstigmasterol via an Oppenauer oxidation followed by ozonolysis andreduction with sodium borohydride.

Alternatively, analogues of compounds of general formulae (I) to (VII)and (IX) to (XII) in which the group at the R⁴ position is —OH may beprepared from an analogue of a compound of general formulae (I) to (VII)and (IX) to (XII) in which R⁴ is OC(O)R¹⁰ by reaction with a base,typically sodium hydroxide as shown in Scheme 3 and Example 1, step C,and Example 7, Scheme 8.

Alcohols with a side chain Y—OH (Y²—CH₂—OH) can be converted tocompounds of general formulae (I) to (VII) and (IX) to (XII) in whichthe side chain is —Y²—C(O)H by oxidation, for example using oxalylchloride suitably in the presence of dimethyl sulfoxide and a base suchas trimethylamine. Alternatively, the oxidation may be carried out usingDess-Martin periodinane as shown in Schemes 3 and 8 of Examples 1 and 6or using sodium hypochlorite.

In compounds of general formulae (I) to (VII) and (IX) to (XII) in whichthe side chain is —Y²—C(O)H, the side chain can be extended, for exampleusing an olefination reaction with a compound of general formula (XVI):Ph₃P═CH—Y³—C(O)OR²⁷  (XVI)

where Y³ is as defined for Y in general formula (I) except that it mayhave a shorter carbon chain such that the linker Y of general formula(I) can be a moiety —Y²—CH₂CH₂—Y³—, wherein Y² and Y³ are as defined forY except that they are shorter in length;

R²⁷ is suitably C₁₋₆ alkyl or benzyl;

To give a compound in which the side chain is Y²—CH═CH—Y³—C(O)OR²⁷.

The olefination reaction may be carried out at about 15 to 25° C.,suitably room temperature, in a solvent such as dichloromethane. Thisreaction is illustrated in Scheme 8 of Example 7.

These compounds can, in turn, be converted to compounds in which R⁴ isthe carboxylic acid mimetic group C(O)NHSO₂R³⁰, wherein R³⁰ is asdefined above, by reaction with:NH₂SO₂R³⁰

wherein R³⁰ is as defined above, in the presence of a coupling agentsuch as 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDCI).

Analogues of compounds of general formulae (I) to (VII) and (IX) to(XII) in which the group at the R⁴ position is OH can be protected witha silyl protecting group. This may be achieved by reaction with acompound of general formula (XV) as described above, typically in anorganic solvent and in the presence of a base, for example imidazole, ortriethylamine. This reaction is shown in Example 1D.

As shown in Scheme 3 of Example 1, analogues of compounds of generalformulae (I) to (VII) and (IX) to (XII) in which the group at the R⁴position is OH may also be converted to analogues of compounds ofgeneral formulae (I) to (VII) and (IX) to (XII) in which the group atthe R⁴ position is a sulfonate, for example methane sulfonate or toluenesulfonate, by reaction with a sulfonyl halide such as methane sulfonylchloride, in the presence of a catalyst such as 4-dimethylaminopyridine(DMAP). Alternatively, they may be converted to compounds of generalformulae (I) to (VII) and (IX) to (XII) in which R⁴ is halo, for examplebromo, by reaction with a halogenating agent, e.g. a brominating agentsuch as carbon tetrabromide as illustrated in Example 1J orN-bromosuccinimide, as illustrated in Example 3A.

Such sulfonate or halide compounds can then be converted to compounds ofgeneral formulae (I) to (VII) and (IX) to (XII) in which R⁴ is cyano byreaction with a cyanide salt, for example sodium or potassium cyanide(see Example 1I and Example 5, Scheme 6). Alternatively, reaction withacetonitrile in the presence of a base such as n-butyl lithium leads toa chain lengthening reaction so that, for example, a side chain —CH₂—O—methanesulfonyl or —CH₂—Br is converted to a side chain —CH₂CH₂—CN (seeExample 4, Scheme 5).

Compounds with a sulfonate or bromide side chain can also be convertedto compounds in which R⁴ is nitro by reaction with nitromethane in thepresence of a base.

Compounds of general formulae (I) to (VII) and (IX) to (XIII) in whichthe side chain is Y²—C(O)OH or an ester thereof may be converted tocompounds in which the side chain is Y²—CH═CH₂ by reaction withPhI(OAc)₂ in the presence of copper (II) acetate using a process similarto Hunsdiecker reaction (see J. Org. Chem., 1986, 51, 404-407 and V. C.Edelsztein et al. Tetrahedron 65 (2009), 3615-3623).

The compounds with side chain —Y²—CH═CH₂ may in turn be oxidised using,for example, osmium tetroxide as described in J. Org. Chem., 1986, 51,404-407 to give a compound in which the side chain is —Y²—CH(OH)—CH₂—OH.Such compounds may be oxidised to compounds in which the side chain isY²—CH(OH)—C(O)H, which may then be protected as a 1,3-dioxane or1,3-dioxolane by reaction with 1,3-propane diol or 1,2-ethandiol in thepresence of an acid catalyst such as toluene sulfonic acid. Similarreactions can be used to prepare the equivalent cyclic thioethers.

Compounds of general formulae (I) to (VII) and (IX) to (XIII) with sidechain —Y—CH═CH₂ may also be prepared by reduction of a compound withside chain —Y≡C—CH, typically by hydrogenation over a palladiumcatalyst, suitably Lindlar catalyst.

Compounds of general formulae (I) to (VII) and (IX) to (XIII) with sidechain —Y—C≡CH may be prepared from compounds with side chain Y—X, whereX is a halo group, particularly bromo, by reaction with anorganometallic reagent, for example:Li—C≡CH.

As described above, compounds of general formulae (I) to (VII) and (IX)to (XIII) wherein R⁴ is halo may be prepared from a correspondingcompound in which R⁴ is OH (for example a compound of general formula(XIV)) by a halogenation reaction. For example, when R⁴ is bromo, thecompound of wherein R⁴ is OH may be reacted with a brominating agentsuch as carbon tetrabromide, N-bromosuccinimide or phosphorustribromide.

Compounds of general formulae (I) to (VII) and (IX) to (XIII) in whichthe side chain —Y—R⁴ is —CH₂—OH may also be converted to compounds inwhich the side chain is ═CH₂. This can be achieved by an eliminationreaction in which the compound having side chain —Y—R⁴ is —CH₂—OH isreacted with an acid such as phosphoric acid, sulphuric acid or toluenesulphonic acid. A similar reaction can be used to convert a compoundwith side chain —Y²—CH₂—OH to a compound with side chain —Y²—C═CH₂.Alternatively, compounds in which the side chain is ═CH₂ can be preparedby oxidising —Y²—CH₂—OH to Y²—CH(O) and then converting this to analkene using an olefination reaction.

Compounds of general formulae (I) to (VII) and (IX) to (XII) with sidechain Y—C≡CH, ═CH₂ or —Y²—C═CH₂ may be reacted with a borane of formula:H—BR¹⁰R¹¹

to give compounds in which the side chain is —Y—CH₂—C(BR¹⁰R¹¹)₂,—CH₂—BR¹⁰R¹¹ or —Y²—CH₂—BR¹⁰R¹¹ respectively.

Compounds of general formulae (I) to (VII) and (IX) to (XII) in whichthe side chain is —CH₂—BR¹⁰R¹¹ or —Y²—CH₂—BR¹⁰R¹¹ may be reacted with,for example phenoxyacetic acid to give a corresponding compound in whichthe side chain is —CH₂—C(O)OH or —Y²—CH₂—C(O)OH.

Compounds of general formulae (I) to (VII) and (IX) to (XII) in which R⁴is —CH[C(O)OR¹⁰]₂ may be prepared from the corresponding compounds ofgeneral formulae (I) to (VII) and (IX) to (XII) in which R⁴ is halo, forexample bromo, by reaction with a malonate ester in the presence of abase such as sodium hydride. A reaction of this type is illustrated inScheme 3 of Example 1 and described in Example 1K for a compound ofgeneral formula (II).

Compounds of general formulae (I) to (VII) and (IX) to (XII) in which R⁴is a malonate ester —CH[C(O)OR¹⁰]₂ may be heated under basic or acidicconditions to give compounds in which R⁴ is CH₂C(O)OH or, when basicconditions are used, a salt thereof. This reaction is shown in Example3, Scheme 4 and Step L.

Compounds of general formulae (I) to (VII) and (IX) to (XIII) in whichthe side chain is —Y—C(O)OH may also be converted to compounds ofgeneral formulae (I) to (VII) and (IX) to (XIII) in which the side chainis —Y—C(O)—CH₂—N₂ by reaction with phosgene to form the acid chloride,followed by reaction with diazomethane.

The diazomethane may be formed in situ using conventional methods, e.g.the treatment of N-nitroso-N-methylurea with aqueous sodium or potassiumhydroxide in diethyl ether. Suitably the diazomethane is used in excess,typically in an amount of greater than 2 equivalents compared with theacid chloride. The reaction is typically conducted in an organic solventsuch as diethyl ether, toluene or a mixture thereof. The reaction iscarried out at a temperature of about −5 to 15° C., typically 0-10° C.

The compound with side chain —Y—C(O)—CH₂—N₂ may be treated with anaqueous silver compound, for example silver nitrate, at an elevatedtemperature and in the presence of an alcohol of formula:R^(10a)—OH

wherein R^(10a) is as defined for R¹⁰ in general formula (I) except thatit is not H. Most suitably, R^(10a) is C₁₋₆ alkyl or benzyl. Under theseconditions, the compound undergoes a Wolff rearrangement to give acompound of general formula (I) to (VII) and (IX) to (XIII) in which theside chain is —Y—CH₂—C(O)OH and thus this sequence can be used tolengthen the side chain.

Compounds of general formulae (I) to (VII) and (IX) to (XIII) in whichthe side chain is Y—C(O)OH, i.e. Y²CH₂CH₂C(O)OH may be converted tocompounds in which the side chain is —Y²—CH₂—CN by reaction with sodiumnitrite under acidic conditions, for example in the presence oftrifluoroacetic acid and trifluroroacetic anhydride (C. D. Schteingartand A. T. Hofmann, Journal of Lipid Research, (1988), 29, 1387-1395;Valentina Sepe et al, Eur. J. Org. Chem. 2012, 5187-5194).

Compounds of general formulae (I) to (VII) and (IX) to (XII) in whichthe side chain is Y—C(O)H may be converted to compounds in which theside chain is —Y—CH(OR¹⁰)(OR¹¹) or —Y—CH(SR¹⁰)(SR¹¹) where R¹⁰ and R¹¹together with the atoms to which they are attached join to form a cyclicgroup. This can be achieved by reacting the compound in which the sidechain is Y—C(O)H with a compound of formula:HX³—(CH₂)_(p)—X³H

where X³ is O or S and p is 2 to 4 but usually is 2 or 3;

or with a protected version of such a compound, for example in which OHor SH groups are protected with trimethylsilyl as shown in Scheme 3,Example 1F and in the first step of Scheme 7 of Example 6.

Compounds of general formulae (I) to (VII) and (IX) to (XII) in whichthe side chain is Y²—C(O)H may also be converted to compounds with sidechain —Y²—CH(OH)—CH₂—CH(OR¹⁰)(OR¹¹), —Y²—CH(OH)—CH₂—CH(R¹⁰)(OR¹¹) or—Y²—CH(OH)—CH₂—CH(SR¹⁰)(SR¹¹) by reaction with an appropriateorganometallic reagent, typically a Grignard reagent of formula:XMg—CH₂—R^(4c);

where X is halo, typically bromo, and R^(4c) —CH(OR¹⁰)(OR¹¹),—CH(R¹⁰)(OR¹¹) or CH(SR¹⁰)(SR¹¹).

An example of this reaction is shown in Scheme 3 of Example 1.

Compounds of general formulae (I) to (VII) and (IX) to (XII) in whichthe side chain is —Y²—CH(OH)—CH₂—CH(R¹⁰)(OR¹¹) can be converted tocompounds in which the side chain is —Y²—CH═CH—C(O)H by reaction with anacid. Following this, the aldehyde can be oxidised to give a carboxylicacid and/or the alkylenene bond can be reduced by hydrogenation to givea saturated side chain in which Y is —Y²—CH₂CH₂—.

Compounds of general formulae (I) to (VII) and (IX) to (XII) in which R⁴is —N₃ may be prepared from analogues of compounds of general formulae(I) to (VII) and (IX) to (XII) in which R⁴ is a leaving group such astoluene sulfonyl, methane sulfonyl or compounds of general formulae (I)to (VII) and (IX) to (XII) in which R⁴ is halo (for example bromo) byreaction with sodium azide. This is illustrated in Example 1G.

Compounds of general formulae (I) to (VII) and (IX) to (XII) in which R⁴is NH₂ may be obtained by reduction of compounds of general formulae (I)to (VII) and (IX) to (XII) in which R⁴ is azide as illustrated inExample 1G.

Compounds of general formulae (I) to (VII) and (IX) to (XII) in which R⁴is —NHC(O)NHSO₂R³⁰ may be prepared from compounds in which R⁴ is NH₂using a coupling reaction with a compound of formula:NH₂SO₂R³⁰

wherein R³⁰ is as defined above;

in the presence of a reagent such as N,N′-carbonyldiimidazole (CDI) orphosgene.

Compounds of general formulae (I) to (VII) and (IX) to (XII) in which R⁴is tetrazol-5-yl may be prepared from compounds of general formulae (I)to (VII) and (IX) to (XII) in which R⁴ is CN by reaction withazidotrimethylsilane/dibutylstannanone or Bu₃SnN₃ as described in US2016/0145295. Alternatively, the compound in which R⁴ is CN may bereacted with sodium azide in the presence of an acid. For example,NaN₃/NH₄Cl in toluene/DMF (Organic and Biomolecular Chemistry, 2008, 6,4108) or NaN₃/NEt₃.HCl in DMF (Brown et al; Bioorg Med Chem Lett, 2002,vol 12, pg 3171). Alternatively, a compound of general formula (I) inwhich R⁴ is azide may be reacted with a suitable cyanide compound, forexample toluene sulfonyl cyanide, under reducing conditions to give acompound in which R⁴ is tetrazol-1-yl.

Compounds of general formulae (I) to (VII) and (IX) to (XII) in which R⁴is amino tetrazole can be prepared from an analogue in which the groupat the R⁴ position is methane sulfonyl by reaction with 5-aminotetrazole.

Compounds of general formulae (I) to (VII) and (IX) to (XIII) in whichthe side chain is —Y²—C(O)H may also be converted to compounds—Y²—CH₂—NR¹⁰R¹¹ by reductive amination, using a reducing agent such as ahydride, borohydride or cyanoborohydride (for example sodiumborohydride, sodium triacetoxyborohydride or sodium cyanoborohydride)and an amine of formula:H—NR¹⁰R¹¹

where R¹⁰ and R¹¹ are as defined above.

Other reactions for modifying the side chains of compounds with asteroid type structure, such as the compounds of general formulae (I) to(VII) and (IX) to (XII), are discussed by Shingate & Hazra, Chem. Rev.2014, 114, 6349-6382, which is incorporated by reference.

An alternative route to analogues of compounds of general formula (III)in which the group at the R⁴ position is an ester is as shown in Scheme1 in which androstenedione is converted to a compound of general formula(V) in which R³ and R⁵ are H; R⁴ is —C(O)OCH₃ and Y is either —CH₂CH₂—or —CH═CH—.

The ester group on the side chain may be converted to a group R⁴ usingthe methods described above.

An alternative route to compounds of general formula (VI) in which Y isan alkenylene group is by use of an olefination reaction, for example aHorner-Wadsworth-Emmons (HWE) olefination of a compound of generalformula (IIA):

which is a compound of general formula (II) in which Y is a bond and R⁴is C(O)H and wherein R³ and R⁵ are as defined for general formula (I);

using a compound of general formula (XVII):

wherein R¹⁰ is as defined for general formula (I).

The reaction may be carried out under standard HWE conditions, forexample using a base such as sodium hydride.

Compounds of general formula (XVII) are readily available or may beprepared by methods known to those of skill in the art.

Other olefination reactions such as a Tebbe olefination, a Wittig typeolefination or a Julia-Kocienski olefination would also give rise tocompounds of general formula (III) in which Y is an alkenylene group.These olefination reactions are familiar to a chemist of skill in theart.

Compounds of general formula (IIA) may be prepared by reaction of acompound of general formula (XVIII) with ozone

wherein R³ and R⁵ are as defined for general formula (I) and R¹⁵ is C₁₋₆alkyl.

An example of a reaction of this type is given in U.S. Pat. No.2,624,748.

Compounds of general formula (XVIII) may be prepared by reaction of acompound of general formula (XIX):

wherein R³ and R⁵ are as defined for general formula (I) and R¹⁵ is C₁₋₆alkyl;

with an acid in a solvent such as methanol.

Compounds of general formula (XIX) may be prepared by oxidation of acompound of general formula (XX):

wherein R³ and R⁵ are as defined for general formula (I) and R¹⁵ is C₁₋₆alkyl using an Oppenauer oxidation.

Examples of the conversion of compounds of general formula (XX) tocompounds of general formula (XVIII) are taught by Shepherd et al, J.Am. Chem. Soc. 1955, 77, 1212-1215 and Goldstein, J. Med. Chem. 1996,39, 5092-5099.

One example of a compound of general formula (XX) is ergosterol, whichis a fungal sterol and Scheme 2 below shows the conversion of ergosterolto a compound similar to general formula (II) in which both R³ and R⁵are H, Y is CH═CH₂ but in which R⁴ is replaced by C(O)OR¹⁰, where R¹⁰ isethyl.

This compound can be converted to a compound of general formula (II) bymodifying the side chain, for example as described above.

Compounds of general formula (I) are synthetic precursors of compoundsof general formula (XXI):

wherein R¹, and Y are as defined in general formula (I);

R^(3a) is H, halo or OH;

R^(4a) is C(O)OR¹⁰, C(O)NR¹⁰R¹¹, S(O)R¹⁰, SO₂R¹⁰, or OSO₂R¹⁰; and

R^(5a) is H or OH.

Compounds of general formula (XXI) are potent agonists of FXR and TGR5and include, in particular, compounds in which R¹ is ethyl. Alsoincluded are the following.

-   -   Compounds in which R⁴ is C(O)OH, for example:        -   obeticholic acid, which is a compound of formula (XXI) in            which R¹ is ethyl, R^(3a) and R^(5a) are both H, Y is            —CH₂CH₂—, and R^(4a) is C(O)OH; and        -   the compound of formula (XXI) in which R¹ is ethyl, R^(3a)            and R^(5a) are both H, Y is —CH₂CH(CH₃)—, and R^(4a) is            C(O)OH; and        -   the compound of formula (XXI) in which R¹ is ethyl, R^(3a)            is H, R^(5a) is OH, Y is —CH₂CH(CH₃)—, and R^(4a) is C(O)OH.    -   Compounds in which R^(4a) is OSO₃H or a salt thereof, for        example:        -   the compound of formula (XXI) in which R¹ is ethyl, R^(3a)            and R^(5a) are both H, Y is —CH₂CH₂—, and R^(4a) is OSO₃H or            a salt thereof; and        -   the compound of formula (XXI) in which R¹ is ethyl, R^(3a)            is H, R^(5a) is OH, Y is —CH₂CH₂CH₂—, and R^(4a) is OSO₃H or            a salt thereof; and        -   the compound of formula (XXI) in which R¹ is ethyl, R^(3a)            is OH, R^(5a) is H, Y is —CH₂CH₂—, and R^(4a) is OSO₃H or a            salt thereof.

Therefore, in a further aspect of the invention there is provided aprocess for the preparation of a compound of general formula (XXI) asdefined above, the process comprising converting a compound of generalformula (I) to a compound of general formula (XXI) by a processcomprising the step of converting the side chain substituent —R⁴ of acompound of general formula (I) to a group R^(4a) as defined above forgeneral formula (XXI).

When R³ and/or R⁵ of the compound of general formula (I) is an OHprotecting group, this protecting group will also be removed at anappropriate stage of the process to give a compound of general formula(XXI) in which R^(3a) and/or R^(5a) is OH.

The conversion of the side chain substituent —R⁴ of a compound ofgeneral formula (I) to a group R^(4a) as defined above for generalformula (XXI) can take place at any stage of the process. For example, acompound of general formula (IA) may be converted to an analogue inwhich the side chain substituent is a group R^(4a) as defined above andthis analogue may be converted in turn to analogues of compounds ofgeneral formula (IB), (IC), (ID) and (IE), which may then be reduced asdescribed above for the conversion of a compound of general formula (IE)to (IF) to obtain a compound of general formula (XXI).

Alternatively, a compound of general formula (IB) may be converted to ananalogue in which the side chain substituent is a group R^(4a) asdefined above and this analogue may be converted in turn to analogues ofcompounds of general formula (IC), (ID) and (IE), which may then bereduced as described above for the conversion of a compound of generalformula (IE) to (IF) to obtain a compound of general formula (XXI).

Alternatively, a compound of general formula (IC) may be converted to ananalogue in which the side chain substituent is a group R^(4a) asdefined above and this analogue may be converted in turn to analogues ofcompounds of general formula (ID) and (IE), which may then be reduced asdescribed above for the conversion of a compound of general formula (IE)to (IF) to obtain a compound of general formula (XXI).

Alternatively, a compound of general formula (ID) may be converted to ananalogue in which the side chain substituent is a group R^(4a) asdefined above and this analogue may be converted to an analogue of thecompound of general formula (IE), which may then be reduced as describedabove for the conversion of a compound of general formula (IE) to (IF)to obtain a compound of general formula (XXI).

Alternatively, a compound of general formula (IE) may be converted to ananalogue in which the side chain substituent is a group R^(4a) asdefined above and this may then be reduced as described above for theconversion of a compound of general formula (IE) to (IF) to obtain acompound of general formula (XXI).

Alternatively, a compound of general formula (IE) may be converted to acompound of general formula (IF) by a process as described above and thecompound of general formula (IF) may be converted to a compound ofgeneral formula (XXI) by converting the side chain substituent —R⁴ to agroup R^(4a) as defined above for general formula (XXI).

For example, compounds of general formula (IF) in which the side chainis —Y²—C(OH)CH₂—CH(OR¹⁰)(OR¹¹), particularly such compounds in which R¹⁰and R¹¹ form a cyclic ether group, may be deprotected to give a compoundin which the side chain is —Y²—C(OH)CH₂—C(O)H. Elimination of watergives a compound with side chain —Y²—CH═CH—C(O)H and this compound canbe oxidised to a compound with side chain —Y²—CH═CH—C(O)OH, which is acompound of general formula (XXI).

Hydrogenation of this compound leads to saturation of the side chaindouble bond, giving a compound of general formula (XXI) in which theside chain is —Y²—CH₂CH₂—C(O)OH.

Compounds of general formula (IF) in which the side chain is —Y²—CH₂—CNmay be hydrolysed to give a compound in which the side chain is—Y²—CH₂—C(O)OH.

Compounds of formula (XXI) can be prepared from other compounds ofgeneral formula (XXI). For example, a compound of general formula (XXI)in which R^(4a) is C(O)OR¹⁰ may be converted to a compound of generalformula (XXI) in which R^(4a) is C(O)NR¹⁰R¹¹, S(O)R¹⁰, SO₂R¹⁰, OSO₂R¹⁰,SO₃R¹⁰, or OSO₃R¹⁰.

Compounds of general formula (XXI) in which R^(4a) is SO₃R¹⁰ may besynthesised from compounds of general formula (XXI) in which R^(4a) isC(O)OH by the methods taught in WO2008/002573, WO2010/014836 andWO2014/066819.

Thus a compound of formula (XXI) in which R^(4a) is C(O)OH may bereacted with a C₁₋₆ alkanoyl or benzoyl chloride or with a C₁₋₆ alkanoicanhydride to protect the OH groups. The protected compound may then bereacted with a reducing agent such as a hydride, suitably lithiumaluminium hydride or sodium borohydride in order to reduce thecarboxylic acid group to OH. The alcohol group may be replaced by ahalogen, for example bromine or iodine, using the triphenylphosphine/imidazole/halogen method described by Classon et al, J. Org.Chem., 1988, 53, 6126-6130. The halogenated compound may then be reactedwith sodium sulphite in an alcoholic solvent to give a compound with aSO₃ ⁻ Na⁺ substituent.

A compound of general formula (XXI) in which R^(4a) is OSO₃R¹⁰ can beobtained by reacting the alcohol obtained from reducing the protectedcarboxylic acid as described above with chlorosulfonic acid in thepresence of a base such as triethylamine to yield the protectedtriethylamine salt. Protecting groups can be removed using basehydrolysis as described above. Reduction of the carboxylic acid followedby reaction of the resultant alcohol with sulfonyl chloride acid yieldsa compound of general formula (XXI) in which R⁴ is OSO₂R¹⁰.

Compounds of general formula (XXI) in which R^(4a) is C(O)NR¹⁰R¹¹ may beprepared from the carboxylic acid by reaction with an amine of formulaH—NR¹⁰R¹¹ in a suitable solvent with heating. Compounds of generalformula (XXI) in which R^(4a) is C(O)NR¹⁰R¹¹ or OSO₃R¹⁰ may also beprepared by methods similar to those described by Festa et al, J. Med.Chem., 2014, 57, 8477-8495.

The methods for modifying the side chains also apply to compounds ofgeneral formulae (I) to (VII) and (IX) to (XIII).

The invention will now be further described with reference to thefollowing examples.

ABBREVIATIONS USED IN EXAMPLES

AcOH Acetic acid Aq. Aqueous nBuLi n-Butyl lithium DCM DichloromethaneDMF N,N-dimethylformamide DMAP 4-Dimethylaminopyridine DMP Dess-Martinperiodinane EtOAc Ethyl acetate EtOH Ethanol EtMgBr Ethyl magnesiumbromide h Hour HFIP 1,1,1,3,3,3-Hexafluoro-2-propanol HMPO(20S)-20-hydroxymethyl-pregna-4-en-3-one also known as20-hydroxymethylpregn-4-en- 3-one and 3-keto-bis-norcholenol IPAIsopropanol mCPBA meta-chloroperoxybenzoic acid MeCN Acetonitrile MeOHMethanol Mesyl Methane sulfonyl MsCl Methane sulfonylchloride MTOMethyltrioxorhenium(VI) NaOMe Sodium methoxide PhMe Toluene PTFEPolytetrafluoroethylene Py Pyridine TBDMSCl tert-Butyldimethylsilylchloride TBME tert-butyl methyl ether TEPA Triethyl phosphonoacetate THFTetrahydrofuran TLC Thin layer chromatography TMSOTf Trimethyl silyltrifluoromethanesulfonate Tosyl Toluene sulfonyl pTSA•H₂O para toluenesulfonic acid monohydrate UHP Urea hydrogen peroxide

Example 1—Preparation of Compounds of General Formula (II)

Scheme 3 below shows the conversion of an analogue of a compound ofgeneral formula (III) in which the side chain is —CH₂OH to analogues ofa compound of general formula (II) in which the side chain is—CH₂OC(O)CH₃ and —CH₂OH and the subsequent conversion of this compoundinto other compounds of general formula (II) with different side chains.

As shown in Scheme 3, the general formula (II) analogue with the —CH₂OHside chain can be converted to compounds of general formula (II) withside chains including —CH₂-9-borabicyclo(3.3.1) nonyl,—CH₂CH₂CH[B(alkyl)₂]₂, —CH₂CN, —CH₂CH₂CN, —CH₂Br, —CH₂CH[C(O)OEt]₂,—CH₂—C≡CH, —CH₂—CH═CH₂, ═CH₂, —C(O)H, —CH₂NH₂,

where X is O or S

alkyl may be C₁₋₆ alkyl and Et is ethyl; and also carboxylic acidmimetic groups including —C(O)NHSO₂R³⁰ and —NHC(O)NH—SO₂R³⁰.

Synthesis of compounds of general formula (II) shown in Scheme 3 isdescribed below.

A. Synthesis of (20S)-20-hydroxymethyl-pregna-4-en-3-one

(20S)-20-Hydroxymethyl-pregna-4-en-3-one (HMPO) can be prepared bychemoselective reduction of dinorcholenaldehyde((20S)-20-formyl-pregn-4-en-3-one) with NaBH₄ in primary alcohol (BarryM. Trost, Alvin C. Lavoie J. Am. Chem. Soc., 1983, 105(15), 5075-5090).

B. Synthesis of (20S)-20-acetoxymethyl-pregna-4,6-dien-3-one

HMPO (300 g, 0.913 mol) was charged to a reaction vessel, followed byAcOH (0.9 L) and toluene (0.3 L) with stirring. p-Chloranil (245 g, 1.00mol) was then charged and the reaction mixture heated to 110° C. andmaintained at this temperature for 6 h. The mixture was then cooled to5° C. and held at that temperature for 2 h. The resulting solid wasfiltered and the filter-cake washed with cold, premixed 3:1 AcOH:Toluene(4×150 mL) and the filtrate was concentrated in-vacuo. The residue wasdissolved in acetone (900 mL), then 3.5% w/w aqueous NaOH (3.0 L) wascharged dropwise with stirring, maintaining the temperature below 30° C.The resulting solids were collected by filtration and the filter cakewas washed with premixed 1:1 acetone:water (1.5 L). The filter cake wasthen slurried in 1:1 acetone:water (600 mL) at 20° C., filtered andwashed with premixed 1:1 acetone:water (1.0 L). The solid was driedunder vacuum at 65-70° C. to give the desired product (224 g, 67%) as atan solid. δH (400 MHz, CDCl₃); 6.17-6.12 (1H, m, C6-CH), 6.10 (1H, dd,J 9.9, 2.0, C7-CH), 5.68 (1H, s, C4-CH), 4.10 (1H, dd, J 10.7, 3.5,C22-CH_(a)H_(b)), 3.79 (1H, dd, J 10.7, 7.4, C22-CH_(a)H_(b)), 2.58 (1H,ddd, J 17.9, 14.4, 5.4, C2-CH_(a)H_(b)), 2.49-2.39 (1H, m,C2-CH_(a)H_(b)), 2.20 (1H, brt, J 10.2, C8-CH), 2.10-1.97 (1H, m), 2.06(3H, s, OC(O)CH₃), 1.96-1.66 (4H, m), 1.62-1.53 (1H, m), 1.52-1.16 (8H,m), 1.12 (3H, s, C19-CH₃), 1.04 (3H, d, J 6.6, C21-CH₃), 0.79 (3H, s,C18-CH₃); δC (100 MHz, CDCl₃); 199.6, 171.3, 163.8, 141.2, 127.9, 123.6,69.4, 53.2, 52.6, 50.7, 43.6, 39.4, 37.7, 36.1, 35.8, 33.9, 33.9, 27.6,23.8, 21.0, 20.7, 17.1, 16.3, 11.9.

C. Synthesis of (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one

(20S)-20-Acetoxymethyl-pregna-4,6-dien-3-one (25 g, 67.5 mmol) wassuspended in MeOH (250 mL) and sodium methoxide (25% w/v solution inMeOH) was added until pH 12 was achieved. The resulting mixture wasstirred at room temperature for 4 h. The pH was adjusted to pH 4 byaddition of Finex CS08GH⁺ resin. The mixture was filtered and thefiltrate was concentrated under reduced pressure, co-evaporating withPhMe (2×250 mL). The residue was dried in a vacuum oven at 30° C. for 48h to give the desired product (22.15 g, 99%) as a light brown solid. δH(400 MHz, CDCl₃); 6.16-6.11 (1H, m, C7-CH), 6.09 (1H, dd, J 9.9, 2.3,C6-CH), 5.67 (1H, s, C4-CH), 3.65 (1H, dd, J 10.5, 3.3,C22-CH_(a)H_(b)), 3.59 (1H, dd, J 10.5, 6.7, C22-CH_(a)H_(b)), 2.57 (1H,ddd, J 18.0, 14.4, 5.5, C2-CH_(a)H_(b)), 2.45-2.38 (1H, m,C2-CH_(a)H_(b)), 2.19 (1H, brt, J 10.4, C8-CH), 2.11-1.76 (5H, m), 1.71(1H, td, J 13.9, 5.3, C1-CH_(a)H_(b)), 1.65-1.16 (9H, m), 1.11 (3H, s,C19-CH₃), 1.06 (3H, d, J 6.6, C21-CH₃), 0.78 (3H, s, C18-CH₃); δC (100MHz, CDCl₃); 199.7, 164.0, 141.4, 127.9, 123.5, 67.8, 53.2, 52.3, 50.7,43.5, 39.4, 38.7, 37.8, 36.1, 33.9, 33.9, 27.6, 23.8, 20.7, 16.7, 16.3,12.0;

D. Synthesis of(20S)-20-tertbutyldimethylsilyloxymethyl-pregna-4,6-dien-3-one

(20S)-20-Hydroxymethyl-pregna-4,6-dien-3-one (1.00 g, 3.04 mmol) wasdissolved in anhydrous CH₂Cl₂ (10 mL) and the solution was cooled to 0°C. Imidazole (414 mg, 6.09 mmol) and TBDMSCI (551 mg, 3.65 mmol) wereadded and the reaction was stirred at 0° C. for 4 h. The reaction waswarmed to room temperature and CH₂Cl₂ (10 mL) and water (20 mL) wereadded. The layers were separated and the organic phase was washed withwater (20 mL), saturated aqueous sodium chloride (20 mL), dried oversodium sulfate and was concentrated under reduced pressure. The residuewas purified by column chromatography on silica gel (0-25% EtOAc inheptane) to give the desired product (890 mg, 66%) as a light yellowsolid. δH (400 MHz, CDCl₃); 6.14 (1H, dd, J 9.9, 1.3, C7-CH), 6.09 (1H,dd, J 9.8, 2.4, C6-CH), 5.66 (1H, s, C4-CH), 3.58 (1H, dd, J 9.7, 3.4,C22-CH_(a)H_(b)), 3.28 (1H, dd, J 9.7, 7.2, C22-CH_(a)H_(b)), 2.57 (1H,ddd, J 17.9, 14.4, 5.4, C2-CH_(a)H_(b)), 2.47-2.37 (1H, m,C2-CH_(a)H_(b)), 2.19 (1H, brt, J 10.3, C8-CH), 2.07 (1H, dt, J 12.9,3.3), 2.00 (1H, dd, J 8.5, 2.1), 1.94-1.63 (3H, m), 1.60-1.15 (9H, m),1.11 (3H, s, C19-CH₃), 1.00 (3H, d, J 6.7, C21-CH₃), 0.89 (9H, s,SiC(CH₃)₃), 0.77 (3H, s, C18-CH₃), 0.03 (6H, s, Si(CH₃)₂); δC (100 MHz,CDCl₃); 199.6, 163.9, 141.5, 127.8, 123.5, 67.7, 53.2, 52.5, 50.7, 43.5,39.4, 39.0, 37.8, 36.1, 34.0, 33.9, 27.6, 25.9, 25.9, 25.9, 23.9, 20.7,18.4, 16.9, 16.3, 12.0, −5.3, −5.4; (IR) v_(max)(cm⁻¹): 3027, 2956,2930, 2891, 2857, 1677, 1077, 753; HRMS (ESI-TOF) m/z: (M+H)⁺ calculatedfor C₂₈H₄₆O₂Si 442.3267, found 443.3338.

E. Synthesis of (20S)-20-formyl-pregna-4,6-dien-3-one

(20S)-20-Hydroxymethyl-pregna-4,6-dien-3-one (3.01 g, 9.16 mmol) wasdissolved in anhydrous CH₂Cl₂ (60 ml) and the solution was cooled to 0°C. Dess-Martin periodinane (5.83 g, 13.7 mmol) was added portion-wiseover 10 minutes and the reaction was allowed to slowly warm to roomtemperature and was stirred for 22 h. The mixture was cooled to 0° C.and a 1:1 mixture of 10% aq. Na₂S₂O₃ and 2% aq. NaHCO₃ (75 ml) was addedportionwise. CH₂Cl₂ (50 mL) was added and the layers were separated. Theaqueous phase was extracted with CH₂Cl₂ (2×50 mL) and the combinedorganics were dried over sodium sulfate and concentrated under reducedpressure. The residue was purified by column chromatography on silicagel (0-25% EtOAc in heptane) to give the desired product (1.23 g, 41%)as a pale yellow solid. δH (400 MHz, CDCl₃); 9.59 (1H, d, J 3.2, CHO),6.12 (2H, s, C6-CH and C7-CH), 5.68 (1H, s, C4-CH), 2.58 (1H, ddd, J17.9, 14.4, 5.4), 2.49-2.36 (2H, m), 2.22 (1H, t, J 10.6, C8-CH),2.08-1.81 (4H, m), 1.73 (1H, td, J 13.8, 5.1, C1-CH_(a)H_(b)), 1.65-1.20(8H, m), 1.15 (3H, d, J 6.9, C21-CH₃), 1.13 (3H, s, C19-CH₃), 0.82 (3H,d, C18-CH₃); δC (100 MHz, CDCl₃); 204.6, 199.5, 163.6, 140.8, 128.1,123.7, 52.8, 50.8, 50.7, 49.4, 44.0, 39.2, 37.6, 36.0, 33.9, 33.9, 27.0,24.1, 20.6, 16.3, 13.5, 12.3; (IR) v_(max)(cm⁻¹): 3030, 2934, 2706,1717, 1655, 1615, 15811; HRMS (ESI-TOF) m/z: (M+H)⁺ calculated forC₂₂H₃₀O₂ 326.2246; found 327.2318.

F. Synthesis of (20S)-20-(ethylenedioxymethyl)-pregna-4,6-dien-3-one

To a solution of (20S)-20-formyl-pregna-4,6-dien-3-one (3.89 g, 12 mmol)in CH₂Cl₂ (5 vol, 20 mL) under an argon atmosphere was added 1,2-bis(trimethylsilyloxy) ethane (2.94 mL, 12 mmol). The reaction mixture wascooled to −78° C. and TMSOTf (108 μL, 0.6 mmol) was added. After 2 h thereaction mixture was diluted with CH₂Cl₂ (100 mL) and washed with water(2×100 mL) and 5% aq. NaCl (100 mL). The organic phase was dried overNa₂SO₄ and was concentrated under reduced pressure. Purification bycolumn chromatography on silica gel gave the desired product (2.42 g,55%) as a colourless crystalline solid. δH (700 MHz, CDCl₃); 6.12 (2H,m), 5.67 (1H, m), 4.86 (1H, d, J 2.0), 3.94 (2H, m), 3.86 (2H, m), 2.56(1H, m), 2.43 (1H, m), 2.19 (1H, t, J 10.6), 2.05-1.95 (3H, m), 1.85 to1.20 (12H, m), 1.11 (3H, s), 0.95 (3H, d, J=6.7), 0.77 (3H, s). δC (176MHz, CDCl₃); 199.7, 163.9, 141.4, 127.9, 123.6, 105.6, 65.3, 65.1, 52.9,52.2, 50.6, 43.7, 39.3, 39.3, 37.8, 36.1, 34.0, 33.9, 27.3, 23.9, 20.67,16.3, 11.7, 11.6.

G. Synthesis of (20S)-20-(1-aminomethyl)-pregna-4,6-dien-3-one

(i) Synthesis of (20S)-tosyloxymethyl-pregna-4,6-dien-3-one

To a solution of (20S)-hydroxymethyl-pregna-4,6-dien-3-one (1.50 g, 4.58mmol) in pyridine (50 mL) at 0° C. was added p-toluenesulfonyl chloride(1.79 g, 9.39 mmol). The reaction was stirred at 0° C. for 1 h andambient for 17 h. The reaction was quenched with 1 M aq. HCl (75 mL) andwas diluted with ethyl acetate (150 mL). The organic phase was separatedand washed with water (50 mL), 5% aq. sodium bicarbonate (75 mL), 5% aq.NaCl (50 mL) and was concentrated in vacuo. The residue was purified bycolumn chromatography on silica gel (heptane-EtOAc) to give the desiredproduct (1.59 g, 72%) as a yellow powder. R_(f): 0.36 (3:2,heptane:ethyl acetate); ¹H NMR (700 MHz, CDCl₃): δ=7.78 (2H, d, J 8.2,Ar—H), 7.35 (2H, d, J 8.2, Ar—H), 6.10 (2H, br. s, C6H and C7H), 5.67(1H, s, C4H), 3.97 (1H, dd, J 9.3, 3.2, C22H), 3.80 (1H, dd, J 9.3, 6.4,C22H), 2.56 (1H, ddd, J 17.6, 14.6, 5.6, C2H), 2.45-2.41 (4H, m, C2H andTs-CH₃), 2.17 (1H, t, J 10.5), 2.01-1.96 (2H, m), 1.80-1.67 (4H, m),1.54 (1H, dq, J 13.5, 3.1), 1.41 (1H, qd, J 13.1, 3.9), 1.30-1.23 (3H,m), 1.23-1.17 (3H, m), 1.10 (3H, s, C19H), 1.00 (3H, d, J 6.7, C21H),0.73 (3H, s, C18H). ¹³C NMR (176 MHz, CDCl₃): δ=197.9, 162.0, 142.9,139.2, 131.3, 128.0, 126.2, 126.1, 121.9, 73.6, 51.3, 49.9, 48.8, 41.7,37.4, 35.9, 34.4, 34.3, 32.2, 32.1, 25.6, 21.9, 20.0, 18.8, 15.1, 14.5,10.1.

(ii) Synthesis of (20S)-azidomethyl-pregna-4,6-dien-3-one

To a suspension of (20S)-tosyloxymethyl-pregna-4,6-dien-3-one (1.58 g,3.27 mmol) in DMF (24 mL) and water (59 μL) was added sodium azide (273mg, 4.20 mmol). The reaction was heated to 70° C. and stirred for 1 h.The reaction was quenched with 2% aq. sodium bicarbonate solution (50mL) at 40° C., and was diluted with ethyl acetate (100 mL). The layerswere separated and the organic layer was washed with 2% aq. sodiumbicarbonate (50 mL), 5% aq. NaCl (50 mL) and was concentrated in vacuo.The residue was purified by column chromatography on silica gel(heptane-EtOAc) to give the desired product (1.01 g, 91% yield) as acolourless crystalline solid. R_(f): 0.54 (3:2, heptane:ethyl acetate);¹H NMR (700 MHz, CDCl₃): δ=6.12 (1H, d, J 9.9, C6H), 6.10 (1H, dd, J9.9, 2.1, C7H), 5.67 (1H, s, C4H), 3.38 (1H, dd, J 11.9, 3.3, C22H),3.07 (1H, dd, J 11.9, 7.3, C22H), 2.57 (1H, ddd, J 17.8, 14.7, 5.4,C2H), 2.46-2.41 (1H, m, C2H), 2.17 (1H, t, J 10.6), 2.04 (1H, dt, J12.8, 3.3), 2.00 (1H, ddd, J 13.2, 5.4, 2.1), 1.93-1.86 (1H, m),1.86-1.81 (1H, m), 1.75-1.65 (2H, m), 1.56 (1H, dq, J 13.4, 3.7), 1.44(1H, qd, J 13.0, 4.0), 1.40-1.28 (6H, m), 1.11 (3H, s, C19H), 1.06 (3H,d, J 6.7, C21H), 0.77 (3H, s, C18H). ¹³C NMR (176 MHz, CDCl₃): δ=199.9,163.8, 141.1, 128.0, 123.6, 57.9, 53.2, 53.0, 50.6, 43.6, 39.3, 37.7,36.9, 36.0, 34.0, 33.9, 27.8, 23.8, 20.6, 17.8, 16.3, 12.0.

(iii) Synthesis of (20S)-aminomethyl-pregna-4,6-dien-3-one

To a solution of (20S)-azidomethyl-pregna-4,6-dien-3-one (99 mg, 0.29mmol) and triphenylphosphine (106 mg, 0.40 mmol) in THF (1.1 mL) underan argon atmosphere, acetone (300 μL) was added. The reaction mixturewas stirred at 18° C. for 64 h. The reaction mixture was diluted withEtOAc (10 mL) and aq. hydrochloric acid solution (10 mL, 2M). The aq.phase was basified with aq. sodium hydroxide solution (6.5 mL, 2M) to pH11, and extracted with EtOAc (10 mL). The organic phase was separatedand concentrated in vacuo. The residue was purified by flashchromatography on silica gel to afford(20S)-aminomethyl-pregna-4,6-dien-3-one as an off-white powder (28 mg,30% yield), R_(f) 0.23 (4:1, CH₂Cl₂:MeOH); ¹H NMR (700 MHz, CDCl₃):δ=6.12-6.07 (2H, m, C6H and C7H), 5.67 (1H, s, C4H), 3.05 (1H, dd, J12.7, 3.1, C22H_(a)H_(b)), 2.74 (1H, dd, J 12.7, 8.3, C22H_(a)H_(b)),2.58 (1H, ddd, J 17.9, 14.5, 5.4, C2H_(a)H_(b)), 2.46-2.41 (1H, m,C2H_(a)H_(b)), 2.18 (1H, t, J 10.5), 2.05-1.94 (3H, m), 1.90-1.81 (2H,m), 1.68 (1H, td, J 13.9, 5.6), 1.55 (1H, dq, J 13.4, 3.4), 1.45-1.17(9H, m), 1.20 (3H, obscured d, J 6.7, C21H), 1.11 (3H, s, C18H), 0.78(3H, s, C19H). ¹³C NMR (140 MHz, CDCl₃): δ=199.5, 163.6, 140.8, 128.0,123.7, 53.2, 52.8, 50.6, 45.3, 43.6, 39.3, 37.6, 36.0, 36.0, 35.1, 34.0,33.9, 27.8, 23.7, 20.7, 17.3, 16.3.

H. Synthesis of (20S)-20-(1-mesyloxymethyl)-pregna-4,6-dien-3-one

To a solution of (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one (1.00 g,3.05 mmol) in pyridine (10 mL) was added DMAP (19 mg, 0.15 mmol). MsCl(1.18 mL, 15.2 mmol) was added dropwise and the reaction was stirred atroom temperature for 18 h. The reaction was cooled in an ice bath andwater (10 mL) was added dropwise. EtOAc (20 mL) was added and the layerswere separated. The aqueous layer was extracted with EtOAc (3×20 mL).The combined organic phases were washed with 2 M aq. HCl (20 mL), driedover sodium sulfate and were concentrated under reduced pressure. Theresidue was purified by column chromatography on silica gel (0-50% EtOAcin heptane) to give the desired product (1.01 g, 82%) as an orangesolid. δH (400 MHz, CDCl₃); 6.12 (2H, brs, C6-CH and C7-CH), 5.68 (1H,s, C4-CH), 4.21 (1H, dd, J 9.4, 3.2, C22-CH_(a)H_(b)), 4.01 (1H, dd, J9.4, 6.6, C22-CH_(a)H_(b)), 3.01 (3H, s, OS(O₂)CH₃), 2.58 (1H, ddd, J18.0, 14.4, 5.5, C2-CH_(a)H_(b)), 2.49-2.39 (1H, m, C2-CH_(a)H_(b)),2.21 (1H, brt, J 10.5, C8-CH), 2.09-1.80 (5H, m), 1.73 (1H, td, J 13.8,5.2, C1-CH_(a)H_(b)), 1.63-1.53 (1H, m), 1.52-1.18 (7H, m), 1.13 (3H, s,C19-CH₃), 1.12 (3H, d, J 6.1, C21-CH₃), 0.80 (3H, s, C18-CH₃); δC (100MHz, CDCl₃); 199.5, 163.6, 140.9, 128.0, 123.7, 74.8, 53.1, 51.8, 50.6,43.6, 39.3, 37.7, 37.2, 36.3, 36.0, 33.9, 33.9, 27.5, 23.8, 20.6, 16.9,16.3, 12.0.

I. Synthesis of (20R)-20-(1-cyanomethyl)-pregna-4,6-dien-3-one

(i) Synthesis of (20S)-20-bromomethyl-4-pregnen-3-one

To a solution of (20S)-hydroxymethyl-4-pregnen-3-one (50 g, 0.15 mol) inCH₂Cl₂ (350 mL) at 0° C. was added triphenylphosphine (43.6 g, 0.17mol). N-bromosuccinimide (29.6 g, 0.17 mol) was added portionwise andthe reaction mixture was stirred at 18° C. After 18 h, the reactionmixture was cooled to 0° C. and triphenylphosphine (19.8 g, 0.08 mol)was added, followed by N-bromosuccinimide (13.5 g, 0.08 mol)portionwise. The mixture was warmed to 18° C. After 2 h the reactionmixture was washed with water (350 mL) and the aqueous phase extractedwith CH₂Cl₂ (350 mL). The combined organic phases were washed with 5%aq. sodium bicarbonate (350 mL), and the aqueous phase extracted withCH₂Cl₂ (100 mL). The combined organic phases were washed with 5% aq.sodium chloride (150 mL), dried over sodium sulfate and wereconcentrated in vacuo. The residue was purified by column chromatographyon silica gel (heptane-EtOAc) to give the desired product (47.1 g, 79%)as a yellow solid. ¹H NMR (700 MHz, CDCl₃): δ=5.72 (1H, s), 3.50 (1H,dd, J=9.8, 2.7, C22-CH_(a)H_(b)), 3.35 (1H, dd, J=9.8, 5.9,C22-CH_(a)H_(b)), 2.45-2.32 (3H, m), 2.27 (1H, ddd, J=14.6, 4.1, 2.5),2.04-1.98 (2H, m), 1.91-1.82 (2H, m), 1.72-1.64 (3H, m), 1.56-1.50 (2H,m), 1.43 (1H, qd, J=13.1, 4.1), 1.33-1.27 (2H, m), 1.22 (1H, dd, J=13.0,4.2), 1.20-1.13 (1H, m), 1.18 (3H, s), 1.09 (3H, d, J=6.4), 1.09-1.00(2H, m), 0.94 (1H, ddd, J=12.3, 10.9, 4.1), 0.74 (3H, s); ¹³C NMR (176MHz, CDCl₃): δ=197.5, 169.3, 121.8, 53.5, 51.6, 51.6, 41.4, 40.4, 37.3,36.5, 35.7, 33.6, 33.6, 31.9, 30.8, 29.9, 25.5, 22.0, 18.9, 16.6, 15.3,10.3.

(ii) Synthesis of (20R)-cyanomethyl-4-pregnen-3-one

To a suspension of (20S)-20-bromomethyl-4-pregnen-3-one (15 g, 38.1mmol) in DMF (225 mL) was added potassium cyanide (7.5 g, 114 mmol). Thesuspension was stirred at 80° C. for 41 h before cooling to roomtemperature. EtOAc (250 mL) and water (500 mL) were added and the layerswere separated. The aqueous layer was extracted with EtOAc (2×250 mL)and the combined organic phases were washed with 5% aq. NaCl (250 mL)and were concentrated under reduced pressure. The residue was purifiedby column chromatography on silica gel (heptane/EtOAc) to afford thedesired product (9.7 g, 75%) as a white solid. δH (700 MHz, CDCl₃); 5.73(1H, s, C4-CH), 2.45-2.32 (4H, m), 2.27 (1H, ddd, J=14.6, 4.2, 2.7),2.24 (1H, dd, J=16.8, 7.1), 2.04-1.99 (2H, m), 1.89-1.78 (3H, m),1.72-1.65 (2H, m), 1.57-1.51 (2H, m), 1.43 (1H, qd, J=13.2, 4.0),1.31-1.16 (4H, m), 1.18 (3H, s), 1.17 (3H, d, J=6.7), 1.11-1.01 (2H, m),0.94 (1H, ddd, J=12.3, 10.7, 4.1), 0.74 (3H, s); δC (176 MHz, CDCl₃);199.5, 171.2, 123.9, 118.9, 55.7, 54.7, 53.6, 42.5, 39.2, 38.5, 35.7,35.6, 34.0, 33.6, 32.8, 31.9, 28.0, 24.8, 24.1, 20.9, 19.3, 17.4, 12.1.

(iii) Synthesis of (20R)-cyanomethyl-4,6-pregnadien-3-one

To a suspension of (20R)-cyanomethyl-4-pregnen-3-one (9.1 g, 26.8 mmol)in toluene (36 mL) and acetic acid (0.15 mL) was added p-chloranil (7.2g, 39.5 mmol). The mixture was heated at reflux for 90 minutes beforeallowing to cool to room temperature. The suspension was filtered,washing with toluene (25 mL). The filtrate was concentrated underreduced pressure and the residue was purified by column chromatographyon silica gel (heptane/EtOAc). The material was then dissolved inacetone (35 mL) and methanol (23 mL) and 0.5 M aq. NaOH (200 mL) wasadded dropwise. Water (100 mL) was added and the resulting solid wasfiltered, washing with water (2×50 mL) and 2:1 acetone:water (2×20 mL).The solid was dried in vacuo to afford the desired product (5.4 g, 60%)as a pale brown solid. δH (700 MHz, CDCl₃); 6.11 (2H, s), 5.67 (1H, s),2.57 (1H, ddd, J=18.0, 14.4, 5.4), 2.45-2.42 (1H, m), 2.37 (1H, dd,J=16.7, 3.7), 2.25 (1H, dd, J=16.7, 7.2), 2.01 (1H, t, J=10.4), 2.03(1H, dt, J=12.8, 3.3), 2.00 (1H, ddd, J=13.2, 5.4, 2.1), 1.96-1.91 (1H,m), 1.88-1.81 (1H, m), 1.74-1.70 (1H, m), 1.58 (1H, dq, J=13.4, 3.6),1.44 (1H, qd, J=4.4, 3.9), 1.36-1.20 (7H, m), 1.18 (3H, d, J=6.7), 1.11(3H, s), 0.79 (3H, s); δC (176 MHz, CDCl₃); 199.6, 163.67, 140.8, 128.1,123.7, 118.8, 54.6, 53.2, 50.5, 43.5, 39.1, 37.6, 36.0, 33.9, 33.9,33.5, 28.0, 24.8, 23.6, 20.6, 19.3, 16.3, 12.0.

J. Synthesis of (20S)-20-(1-bromomethyl)-pregna-4,6-dien-3-one

To a solution of (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one (1.00 g,3.05 mmol) in anhydrous CH₂Cl₂ (10 mL) was added carbon tetrabromide(1.52 g, 4.57 mmol). Triphenylphosphine (1.20 g, 4.57 mmol) was addedand the mixture was heated at reflux for 2 h. The reaction was allowedto cool to room temperature and water (20 mL) was added. The layers wereseparated and the organic layer was washed with 5% aq. NaHCO₃ (20 mL),10% aq NaCl (20 mL) and was concentrated under reduced pressure. Theresidue was purified by column chromatography on silica gel (0-25%acetone in heptane) to give the desired product (980 mg, 82%) as a lightyellow crystalline solid. δH (400 MHz, CDCl₃); 6.09-6.00 (2H, m, C6-CHand C7 CH), 5.59 (1H, s, C4-CH), 3.43 (1H, dd, J 9.8, 2.7,C22-CH_(a)H_(b)), 3.29 (1H, dd, J 9.8, 5.8, C22-CH_(a)H_(b)), 2.50 (1H,ddd, J 17.9, 14.4, 5.4, C2-CH_(a)H_(b)), 2.40-2.30 (1H, m,C2-CH_(a)H_(b)), 2.13 (1H, brt, J 9.8, C8-CH), 2.01-1.57 (5H, m),1.55-1.45 (1H, m), 1.44-1.10 (8H, m), 1.05 (3H, s, C19-CH₃), 1.03 (3H,d, J 6.5, C21-CH₃), 0.72 (3H, s, C18-CH₃); δC (100 MHz, CDCl₃); 199.2,163.6, 141.0, 127.9, 123.6, 53.5, 53.1, 50.6, 43.4, 43.3, 39.2, 37.7,37.6, 36.0, 33.9, 33.9, 27.4, 23.6, 20.6, 18.6, 16.3, 12.3.

K. Synthesis of 23-ethoxyformyl-3-oxo-4,6-choladien-24-oic Acid EthylEster

Sodium hydride (60% dispersion in mineral oil, 226 mg, 5.64 mmol) wassuspended in anhydrous THF (10 mL) and the mixture was cooled to 0° C.Diethyl malonate (1.17 mL, 7.68 mmol) was added drop-wise and themixture was stirred at 0° C. for 15 minutes. A solution of(20S)-20-(bromomethyl)-pregna-4,6-dien-3-one (1.00 g, 2.56 mmol) inanhydrous THF (10 mL) was added drop-wise and the reaction was heated atreflux for 18 h. The reaction was allowed to cool to room temperatureand water (10 mL) was added. EtOAc (25 mL) was added and the layers wereseparated. The aqueous layer was extracted with EtOAc (3×50 mL) and thecombined organics were washed with 10% aq. NaCl (50 mL), dried oversodium sulfate and were concentrated under reduced pressure. The residuewas purified by column chromatography on silica gel (0-25% acetone inheptane) to give the desired product (1.00 g, 83%) as a clear oil. δH(400 MHz, CDCl₃); 6.17-6.07 (2H, m, C6-CH and C7-CH), 5.67 (1H, s,C4-CH), 4.29-4.14 (4H, m, 2×C(O)OCH₂), 3.44 (1H, dd, J 10.9, 3.7,EtO₂CCH), 2.57 (1H, ddd, J 17.9, 14.4, 5.4, C2-CH_(a)H_(b)), 2.43 (1H,dddd, J 17.8, 5.1, 2.0, 0.8, C2-CH_(a)H_(b)), 2.24-2.12 (2H, m),2.10-1.93 (3H, m), 1.87-1.77 (1H, m), 1.71 (1H, td, J 16.2, 5.2,C1-CH_(a)H_(b)), 1.59-1.35 (4H, m), 1.34-1.14 (12H, m), 1.11 (3H, s,C18-CH₃), 0.96 (3H, d, J 6.2, C21-CH₃), 0.75 (3H, s, C19-CH₃); δC (100MHz, CDCl₃); 199.5, 170.0, 169.6, 163.8, 141.3, 127.9, 123.6, 61.4,61.2, 56.2, 53.4, 50.6, 49.8, 43.5, 39.5, 37.7, 36.1, 35.0, 34.3, 34.0,33.9, 28.0, 23.7, 20.7, 18.2, 16.3, 14.2, 14.1, 11.9.

L. Synthesis of(20S)-20-(5-Tosyltetrazol-1-yl)methyl-pregna-4,6-dien-3-one

To a solution of (20S)-azidomethyl-pregna-4,6-dien-3-one (500 mg, 1.41mmol) in CH₂Cl₂ (5 mL) was added p-toluenesulfonyl cyanide (282 mg, 1.55mmol). Copper(I) trifluoromethanesulfonate benzene complex (71 mg, 0.141mmol) was added and the mixture was stirred at room temperature for 18h. Toluene (5 mL), added p-toluenesulfonyl cyanide (128 mg, 0.708 mmol)and copper(I) trifluoromethanesulfonate benzene complex (71 mg, 0.141mmol) were added and the mixture was heated to 60° C. for 24 h. Water(10 mL) and CH₂Cl₂ (30 mL) were added and the layers were separated. Theorganic layer was washed with 10% aq. Na₂S₂O₃/2% aq. NaHCO₃ (2×20 mL),10% aq. NaCl (20 mL), was dried over sodium sulfate and was concentratedunder reduced pressure. The residue was purified by columnchromatography on silica gel (0-50% EtOAc in heptane) to give thedesired product (381 mg, 50%) as a light yellow solid. δH (400 MHz,CDCl₃); 8.03-7.97 (2H, m, ArH), 7.46 (2H, m, ArH), 6.14 (2H, brs, C6-CHand C7-CH), 5.69 (1H, s, C4-CH), 4.80 (1H, dd, J 13.4, 3.9,C22-CH_(a)H_(b)), 4.45 (1H, dd, J 13.4, 10.5, C22-CH_(a)H_(b)),2.26-2.53 (1H, m), 2.51 (3H, s, ArCH₃), 2.49-2.28 (2H, m), 2.24 (1H,appt, J, 10.5), 2.13-1.97 (2H, m), 1.96-1.87 (1H, m), 1.79-1.63 (2H, m),1.53-1.18 (8H, m), 1.13 (3H, s, C19-CH₃), 0.89 (3H, d, J 6.6, C21-CH₃),0.86 (3H, s, C18-CH₃); δC (100 MHz, CDCl₃); 199.5, 163.6, 147.5, 140.8,134.3, 130.4, 129.3, 128.1, 123.7, 55.1, 53.9, 53.2, 50.7, 44.0, 39.4,37.8, 37.6, 36.0, 33.9, 33.9, 31.9, 27.5, 23.8, 22.7, 21.9, 20.6, 16.5,16.3, 12.0.

M. Synthesis of N-((22E)-3,24-dioxo-4,6,22-cholatrien-24-yl)cyclopropylSulfonamide

(i) Synthesis of (22E)-3-Oxo-4,6,22-cholatrien-24-oic Acid

(22E)-3-Oxo-4,6,22-cholatrien-24-oic acid ethyl ester (10 g, 25.2 mmol)was suspended in IPA (100 mL) and the mixture was heated to 60° C. 0.5 Maq. NaOH (60 mL, 30 mmol) was added and the mixture was stirred at 60°C. for 3 h. The volatiles were removed under reduced pressure and EtOAc(250 mL) was added. The mixture was acidified to pH 1 using 2 M aq. HCl,and further EtOAc (100 mL) was added. The layers were separated and theorganic layer was washed with water (3×100 mL) and concentrated underreduced pressure. The residue was dissolved in EtOAc (200 mL) withheating and was then cooled to −20° C. for 18 h. The solid formed wasfiltered, washing with EtOAc (20 mL). The solid was then dried underreduced pressure to give the desired product (4.55 g, 49%) as a tansolid. δH (400 MHz, CDCl₃); 6.94 (1H, dd, J 15.6, 9.0, C23-CH), 6.11(2H, brs, C6-CH and C7-CH), 5.77 (1H, dd, J 15.6, 0.6, C22-CH), 5.68(1H, s, C4-CH), 2.58 (1H, ddd, J 18.0, 14.4, 5.4, C2-CH_(a)H_(b)),2.51-2.40 (1H, m, C2-CH_(a)H_(b)), 2.40-2.28 (1H, m), 2.21 (1H, appt, J10.1), 2.10-1.95 (2H, m), 1.89-1.65 (3H, m), 1.64-1.53 (1H, m),1.53-1.39 (1H, m), 1.38-1.18 (7H, m), 1.12 (3H, s, C19-CH₃), 1.12 (3H,d, J 6.6, C21-CH₃), 0.81 (3H, s, C18-CH₃); δC (100 MHz, CDCl₃); 199.7,171.8, 163.9, 156.9, 141.1, 128.0, 123.6, 118.6, 54.7, 53.2, 50.7, 43.7,39.7, 39.3, 37.7, 36.1, 33.9, 33.9, 27.8, 23.7, 20.6, 19.1, 16.3, 12.1.

(ii) Synthesis ofN-((22E)-3,24-dioxo-4,6,22-cholatrien-24-yl)cyclopropylsulfonamide

To a solution of (22E)-3-oxo-4,6,22-cholatrien-24-oic acid (2.00 g, 5.43mmol) in CH₂Cl₂ (40 mL) was added EDCI (1.69 g, 10.9 mmol) and DMAP(1.33 g, 10.9 mmol). Cyclopropane sulfonamide (1.97 g, 16.3 mmol) wasadded and the reaction was stirred at room temperature for 22 h. Water(25 mL) was added and the layers were separated. The aqueous layer wasextracted with CH₂Cl₂ (2×25 mL) and the combined organics were washedwith 2 M aq HCl (20 mL), 10% aq. NaCl (10 mL), dried over sodium sulfateand concentrated under reduced pressure. The residue was purified bycolumn chromatography on silica gel (0-10% acetone in toluene) to givethe desired product (1.68 g, 66%) as an off-white solid. δH (400 MHz,CDCl₃); 8.90 (1H, s, NH), 6.95 (1H, dd, J 15.5, 9.0, C23-CH), 6.11 (2H,brs, C6-CH and C7-CH), 5.86 (1H, dd, J 15.5, 0.5, C22-CH), 5.68 (1H, s,C4-CH), 3.00 (1H, dddd, J 12.8, 9.5, 8.1, 4.8, SO₂CH), 2.64 (1H, ddd, J18.1, 14.4, 5.4, C2-CH_(a)H_(b)), 2.51-2.41 (1H, m, C2-CH_(a)H_(b)),2.40-2.28 (1H, m), 2.25-2.15 (1H, m), 2.09-1.96 (2H, m), 1.85-1.64 (3H,m), 1.63-1.52 (1H, m), 1.51-1.17 (9H, m), 1.17-1.07 (5H, m), 1.12 (3H,s, C19-CH₃), 0.80 (3H, s, C18-CH₃); δC (100 MHz, CDCl₃); 200.0, 164.2,164.1, 155.5, 141.3, 127.9, 123.6, 119.4, 54.7, 53.2, 50.6, 43.8, 39.8,39.3, 37.8, 36.1, 33.9, 33.9, 31.5, 28.1, 23.7, 20.6, 19.1, 16.3, 12.2,6.3, 6.3.

N. Synthesis ofN-((22E)-3,24-dioxo-4,6,22-cholatrien-24-yl)-4-(trifluoromethoxy)benzenesulfonamide

To a solution of (22E)-3-oxo-4,6,22-cholatrien-24-oic acid (2.00 g, 5.43mmol) in CH₂Cl₂ (40 mL) was added EDCI (1.69 g, 10.9 mmol) and DMAP(1.33 g, 10.9 mmol). 4-(Trifluoromethoxy)benzene sulfonamide (3.93 g,16.3 mmol) was added and the reaction was stirred at room temperaturefor 22 h. Water (25 mL) was added and the layers were separated. Theaqueous layer was extracted with CH₂Cl₂ (2×25 mL) and the combinedorganics were washed with 2 M aq HCl (20 mL), 10% aq. NaCl (10 mL),dried over sodium sulfate and concentrated under reduced pressure. Theresidue was used in the next step without purification. A portion waspurified by column chromatography on silica gel (0-50% EtOAc in heptane)to give the desired product as an off-white solid. δH (400 MHz, MeOD);8.16-8.11 (2H, m, ArH), 7.52-7.46 (2H, m, ArH), 6.82 (1H, dd, J 15.4,9.0, C23-CH), 6.20 (1H, brdd, J 9.8, 1.4, C6-CH), 6.15 (1H, dd, J 9.9,1.4, C7-CH), 5.82 (1H, dd, J 15.4, 0.7, C22-CH), 5.64 (1H, s, C4-CH),2.62 (1H, ddd, J 18.2, 14.5, 5.4, C2-CH_(a)H_(b)), 2.42-2.20 (3H, m),2.12-1.98 (2H, m), 1.88-1.63 (3H, m), 1.63-1.55 (1H, m), 1.49 (1H, dd, J12.6, 3.8), 1.40-1.18 (7H, m), 1.14 (3H, s, C19-CH₃), 1.08 (3H, d, J6.6, C21-CH₃), 0.81 (3H, s, C18-CH₃); δC (100 MHz, MeOD); 202.3, 167.2,165.9, 156.7, 154.0, 143.3, 139.7, 131.8, 128.8, 123.9, 123.0 (q, J254), 121.9, 120.6, 56.0, 54.6, 52.2, 44.9, 40.9, 40.6, 39.1, 37.4,35.0, 34.7, 30.2, 29.0, 24.7, 21.7, 19.5, 16.6, 12.5.

O. Synthesis of (20S)—(N-benzyl)aminomethyl-pregna-4,6-dien-3-one

(20S)-Formyl-pregna-4,6-dien-3-one (98 mg, 0.30 mmol) and benzylamine(21 μL, 0.30 mmol) were dissolved in 1,2-dichloroethane (1.0 mL) underan argon atmosphere. Sodium triacetoxyborohydride (96 mg, 0.45 mmol) wasadded. The reaction mixture was stirred at 20° C. for 2 h, then quenchedwith aq. sodium bicarbonate solution (5%, 2 mL). The mixture was dilutedwith EtOAc (10 mL) and water (5 mL). The aq. phase was separated andextracted with EtOAc (2×5 mL). The organic phases were combined andconcentrated in vacuo. The residue was purified by silica columnchromatography (heptane-EtOAc) to yield(20S)—(N-benzyl)aminomethyl-pregna-4,6-dien-3-one as a beige powder (51mg, 41% yield). R_(f) 0.15 (EtOAc); ¹H NMR (500 MHz, CDCl₃): δ=7.34 (4H,d, J 4.5, Bn-CH), 7.29-7.23 (1H, m, Bn-CH), 6.15 (1H, d, J 10.2, C6),6.11 (1H, dd, J 9.6, 2.0, C7H), 5.68 (1H, s, C4H), 3.84 (1H, d, J 13.1,Bn-CH_(a)H_(b)), 3.75 (1H, d, J 13.1, Bn-CH_(a)H_(b)), 2.69 (1H, dd, J11.6, 3.0, C22H_(a)H_(b)), 2.58 (1H, ddd, J 17.2, 14.5, 5.3,C2H_(a)H_(b)), 2.44 (1H, dd, J 17.4, 4.4, C2H_(a)H_(b)), 2.35 (1H, dd, J11.5, 8.3, C22H_(a)H_(b)), 2.20 (1H, t, J 10.7, H8), 2.07 (1H, dt, J12.6, 3.0), 2.04-1.97 (1H, m, C1H_(a)H_(b)), 1.92-1.68 (3H, m),1.68-1.60 (1H, m, C20H), 1.60-1.52 (1H, m), 1.44 (1H, qd, J 12.8, 3.9),1.40-1.18 (7H, m), 1.13 (3H, s, C18H), 1.04 (3H, d, J 6.6, C21H), 0.78(3H, s, C19H). ¹³C NMR (126 MHz, CDCl₃): δ=199.7, 164.0, 141.4, 140.5,128.4, 128.1, 127.8, 126.9, 123.5, 54.9, 54.2, 54.0, 53.3, 50.7, 43.5,39.5, 37.7, 36.5, 36.0, 34.0, 33.9, 27.9, 23.8, 20.7, 17.8, 16.3, 12.0.

Example 2—Preparation of Compounds of General formula (IA) A.Epoxidation of (22E)-3-oxo-4,6,22-cholatrien-24-oic Acid Ethyl EsterUsing Methyltrioxorhenium to Form (6α, 7α,22E)-6,7-epoxy-3-oxo-4,22-choladien-24-oic Acid Ethyl Ester

To a solution of (22E)-3-oxo-4,6,22-cholatrien-24-oic acid ethyl ester(5.00 g, 12.6 mmol) in HFIP (20 mL, 4 volumes) and EtOAc (10 mL, 2volumes) was added MTO (37 mg, 0.126 mmol) and 3-methylpyrazole (122 μl,1.51 mmol) and the mixture was cooled to 5° C. UHP (1.30 g, 13.9 mmol)was added portion-wise and the mixture was stirred at 5° C. for 24 h.After 24 h, a second addition of MTO (37 mg, 0.126 mmol) and UHP (1.30g, 13.9 mmol) was conducted and the reaction was stirred at 5° C. for 18h. The reaction was then quenched by the portion-wise addition of 12%aq. NaHSO₃ (15 mL) maintaining the temperature <25° C. The mixturestirred for 0.5 h whilst warming to ambient temperature, to ensure allperoxide was quenched. Water (12.5 mL) and EtOAc (5 mL) were added andthe layers separated. The organic phase was washed with 5% aq. NaHCO₃(20 mL), water (20 mL) and then concentrated under reduced pressure. Thecrude material (5.72 g) was crystallised from EtOAc (15 mL).

Further Epoxidation Reactions of Compounds of Formula (II)

General Procedure A: MTO Catalyzed Epoxidation

To a solution of a compound of general formula (II) (1 eq.) and MTO (1mol %) in EtOAc (2 vol) and HFIP (4 vol) was added 3-methylpyrazole(0.12 eq.) and the mixture was cooled to 5° C. UHP (1.1 eq) was addedand the mixture was stirred for 18-50 h until deemed complete by TLCanalysis. The reaction mixture was then quenched with the addition of12% aq. NaHSO₃ (3 vol) then partitioned between water (2.5 vol) andEtOAc (1 vol). The phases were separated and the organic phase washedwith 5% aq. NaHCO₃ (4 vol) and water (4 vol). After concentration underreduced pressure the crude residue was purified by column chromatography(SiO₂, eluting with heptane:EtOAc gradient).

B. Epoxidation of (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one to Form(6α, 7α, 20S)-6,7-epoxy-20-hydroxymethyl-pregn-4-en-3-one

(20S)-20-hydroxymethyl-pregna-4,6-dien-3-one (500 mg, 1.52 mmol) wasepoxidized using MTO according to the General Procedure A to yield thetitle compound (210 mg, 40%) as a light yellow solid.

δH (400 MHz, CDCl₃); 6.11 (1H, s, C4-CH), 3.66 (1H, dd, J 10.4, 3.3,C22-CH_(a)H_(b)), 3.45 (1H, d, J 3.7, C6-CH), 3.42-3.32 (2H, m, C7-CHand C22-CH_(a)H_(b)), 2.56 (1H, ddd, J 18.2, 14.1, 5.5, C2-CH_(a)H_(b)),2.45 (1H, dddd, J 18.0, 5.3, 2.0, 0.8, C2-CH_(a)H_(b)), 2.02 (1H, dt, J12.8, 2.7, C12-CH_(a)H_(b)), 1.98-1.83 (4H, m), 1.71 (1H, td, J 13.6,5.5, C1-CH_(a)H_(b)), 1.65-1.16 (10H, m), 1.10 (3H, s, C19-CH₃), 1.06(3H, d, J 6.6, C21-CH₃), 0.77 (3H, s, C18-CH₃); δC (100 MHz, CDCl₃);198.3, 162.7, 131.1, 67.8, 54.6, 52.5, 52.5, 51.1, 43.2, 40.6, 39.2,38.8, 35.6, 34.7, 34.1, 33.9, 27.8, 23.8, 19.9, 17.2, 16.7, 11.9.

C. Epoxidation of (20S)-20-(1-bromomethyl)-pregna-4,6-dien-3-one to Form(6α, 7α, 20S)-20-(1-bromomethyl-6,7-epoxy-pregn-4-en-3-one

(20S)-20-(1-bromomethyl)-pregna-4,6-dien-3-one (500 mg, 1.28 mmol) wasepoxidized using MTO according to General Procedure A to yield the titlecompound (290 mg, 56%) as a light brown solid.

δH (400 MHz, CDCl₃); 6.12 (1H, s, C4-CH), 3.52 (1H, dd, J 9.8, 2.6,C22-CH_(a)H_(b)), 3.46 (1H, d, J 3.7, C6-CH), 3.39-3.17 (2H, m, C7-CHand C22-CH_(a)H_(b)), 2.56 (1H, ddd, J 18.1, 14.0, 5.4, C2-CH_(a)H_(b)),2.47 (1H, dddd, J 18.0, 5.5, 2.2, 0.9, C2-CH_(a)H_(b)), 2.05-1.84 (5H,m), 1.79-1.66 (2H, m), 1.58-1.46 (1H, m), 1.44-1.19 (7H, m), 1.11 (3H,d, J 6.3, C21-CH₃), 1.10 (3H, s, C19-CH₃), 0.78 (3H, s, C18-CH₃); δC(100 MHz, CDCl₃); 198.2, 162.6, 131.2, 54.5, 53.5, 52.5, 51.2, 43.1,43.0, 40.6, 39.0, 37.8, 35.6, 34.7, 34.1, 33.9, 27.6, 34.6, 19.9, 18.6,17.2, 12.2.

D. Epoxidation of (20S)-20-(1-mesyloxymethyl)-pregna-4,6-dien-3-one toForm (6α, 7α, 20S)-20-(1-mesyloxymethyl)-6,7-epoxy-pregn-4-en-3-one

(20S)-20-(1-mesyloxymethyl)-pregna-4,6-dien-3-one (500 mg, 1.24 mmol)was epoxidized using MTO according to General Procedure A to yield thetitle compound (460 mg, 88%) as a light yellow solid.

δH (400 MHz, CDCl₃); 6.12 (1H, s, C4-CH), 4.22 (1H, dd, J 9.4, 3.2,C22-CH_(a)H_(b)), 3.99 (1H, dd, J 9.4, 6.9, C22-CH_(a)H_(b)), 3.46 (1H,brd, J 3.7, C6-CH), 3.34 (1H, brd, J 3.6, C7-CH), 3.01 (3H, s,OS(O₂)CH₃), 2.56 (1H, ddd, J 18.2, 14.1, 5.5, C2-CH_(a)H_(b)), 2.50-2.41(1H, m), 2.05-1.80 (6H, m), 1.72 (1H, td, J 13.6, 5.6, C1-CH_(a)H_(b)),1.65-1.17 (8H, m), 1.11 (3H, d, J 6.5, C21-CH₃), 1.10 (3H, C19-CH₃),0.76 (3H, s, C18-CH₃); δC (100 MHz, CDCl₃); 198.2, 162.5, 131.2, 74.7,54.5, 52.5, 51.8, 51.1, 43.3, 40.6, 39.1, 37.3, 36.4, 35.6, 34.7, 34.1,33.9, 27.7, 23.7, 19.9, 17.2, 16.8, 11.9.

E. Epoxidation of(20S)-20-(1-tertbutyldimethylsilyloxymethyl)-pregna-4,6-dien-3-one toform (6α, 7α,20S)-20-(1-tert-butyldimethylsilyloxymethyl)-6,7-epoxy-pregn-4-en-3-one

(20S)-20-(1-tertbutyldimethylsilyloxymethyl)-pregna-4,6-dien-3-one (500mg, 1.13 mmol) was epoxidized using MTO according to General Procedure Ato yield the title compound (100 mg, 19%) as a light brown solid.

δH (400 MHz, CDCl₃); 6.11 (1H, s, C4-CH), 3.58 (1H, dd, J 9.6, 3.3,C22-CH_(a)H_(b)), 3.45 (1H, d, J 3.7, C6-CH), 3.42 (1H, brd, J 3.5,C7-CH), 3.28 (1H, dd, J 9.6, 7.2, C22-CH_(a)H_(b)), 2.55 (1H, ddd, J18.2, 14.1, 5.5, C2-CH_(a)H_(b)), 2.49-2.40 (1H, m, C2-CH_(a)H_(b)),2.02 (1H, td, J 12.8, 3.0, C12-CH_(a)H_(b)), 1.98-1.82 (4H, m), 1.71(1H, td, J 13.6, 5.5, C1-CH_(a)H_(b)), 1.61-1.14 (9H, m), 1.10 (3H, s,C19-CH₃), 1.00 (3H, d, J 6.6, C21-CH₃), 0.89 (9H, s, SiC(CH₃)₃), 0.75(3H, s, C18-CH₃), 0.06 (6H, d, J 0.6, 2×SiCH₃); δC (100 MHz, CDCl₃);198.3, 162.8, 131.1, 67.7, 54.7, 52.6, 52.3, 51.1, 43.1, 40.7, 39.2,39.0, 35.6, 34.7, 34.1, 33.9, 27.8, 26.0, 26.0, 26.0, 23.8, 19.9, 18.4,17.2, 16.9, 11.9, −5.3, −5.4.

F. Epoxidation of (20S)-20-acetoxymethyl-pregna-4,6-dien-3-one to Form(6α, 7α, 20S)-20-acetoxymethyl-6,7-epoxy-pregn-4-en-3-one

The product was prepared according to the general procedure for MTOcatalysed epoxidation on 200 g scale, isolated in 50% yield (105 g) as atan solid.

¹H NMR (700 MHz, CDCl₃): δ=6.11 (1H, s), 4.09 (1H, dd, J 10.7, 3.4),3.79 (1H, dd, J 10.7, 7.4), 3.45 (1H, d, J 3.7), 3.34 (1H, d, J 3.5),2.55 (1H, m), 2.46 (1H, m), 2.05 (3H, s), 2.02-1.85 (5H, m), 1.78-1.68(2H, m), 1.55-1.20 (8H, m), 1.10 (3H, s), 1.02 (3H, d, J 6.6), 0.76 (3H,s); ¹³C NMR (175 MHz, CDCl₃): δ=198.3, 171.3, 162.7, 131.1, 69.3, 54.6,52.5, 52.4, 51.1, 43.2, 40.6, 39.1, 35.8, 35.6, 34.6, 34.1, 33.9, 27.7,23.7, 21.0, 19.9, 17.2, 17.1, 11.8.

G. Epoxidation of (20S)-20-(ethylenedioxymethyl)-pregna-4,6-dien-3-one(Example 1F) to Form (6α, 7α,20S)-6,7-epoxy-20-(ethylenedioxymethyl)-pregn-4-en-3-one

(20S)-20-(ethylenedioxymethyl)-pregna-4,6-dien-3-one (3.15 g, 8.5 mmol)and BHT (57 mg, 0.26 mmol) were charged to a flask under argon, followedby EtOAc (8 vol, 25 mL) and water (2.5 vol, 7.9 mL) and the mixtureheated to 80° C. mCPBA 70% (3.69 g, 15 mmol) in EtOAc (5 vol, 16 mL) wasadded dropwise over 10 minutes and the reaction mixture then stirred at70° C. for 1 h (TLC, eluant 1:1 EtOAc:Heptane; visualized with CeriumAmmonium Molybdate stain). The reaction mixture was allowed to cool toroom temperature and washed with 1M aq. NaOH (3×50 mL) and 10% aq.Na₂S₂O₃ (3×50 mL). After a negative test for peroxides the organic phasewas dried over Na₂SO₄ and concentrated in-vacuo at 40° C. Purificationby column chromatography and concentration in-vacuo at 40° C. gave (6α,7α, 20S)-6,7-epoxy-20-(ethylenedioxymethyl)-pregna-4-en-3-one as a whitecrystalline solid (1.15 g). ¹H NMR (700 MHz, CDCl₃): δ=6.31 (1H, s),4.85 (1H, d, J 2.0), 4.0-3.8 (2H, m), 3.45 (1H, d, J 3.7), 3.35 (1H, d,J 3.6), 2.59-2.43 (2H, m), 2.05-1.68 (8H, m), 1.55-1.20 (10H, m), 1.10(3H, s), 0.93 (3H, d, J 6.6), 0.75 (3H, s). ¹³C NMR (176 MHz, CDCl₃):δ=198.6, 163.0, 131.0, 105.9, 65.2, 65.0, 54.7, 52.5, 51.9, 50.8, 43.4,40.6, 39.3, 39.0, 35.6, 34.6, 34.1, 33.8, 27.4, 23.8, 19.9, 17.2, 11.6,11.6.

H. Epoxidation of (20S)-azidomethyl-pregna-4,6-dien-3-one to form (6α,7α, 20S)-6,7-epoxy-20-azidomethyl-pregna-4-en-3-one

To a solution of (20S)-azidomethyl-pregna-4,6-dien-3-one (203 mg, 0.598mmol) and 3-methylpyrazole (3 μL, 0.04 mmol) in HFIP (0.8 mL) underargon atmosphere at 10° C., MTO (3.2 mg, 0.013 mmol) and UHP (64 mg,0.68 mmol) were added. The reaction was stirred at 10° C. for 2 h, andquenched with 5% aq. sodium bisulfite solution (1.0 mL). The reactionwas diluted with ethyl acetate (10 mL) and washed with water (10 mL) and10% aq. sodium bicarbonate solution (10 mL). The organic phase wasseparated and concentrated in vacuo. The residue was purified by columnchromatography on silica gel (heptane-EtOAc, R_(f) in 3:2heptane:EtOAc=0.42) to the desired product (99 mg, 47%) as a whitepowder. ¹H NMR (700 MHz, CDCl₃): δ=6.11 (1H, s, C4-CH), 3.46 (1H, d,J=3.7, C6-CH), 3.39 (1H, dd, J=11.9, 3.3, C22-CH_(a)H_(b)), 3.34 (1H, d,J=3.7, C7-CH), 3.06 (1H, dd, J=11.9, 7.5, C22-CH_(a)H_(b)), 2.55 (1H,ddd, J=18.0, 14.3, 5.5, C2-CH_(a)H_(b)), 2.48-2.44 (1H, m,C2-CH_(a)H_(b)), 2.00 (1H, dt, J=11.9, 3.3), 1.97-1.90 (3H, m), 1.87(1H, td, J=10.8, 1.4, C8-CH), 1.74-1.63 (2H, m), 1.53 (1H, dq, J=12.7,3.5), 1.49-1.45 (1H, m), 1.41-1.23 (5H, m), 1.22 (1H, td, J=12.7, 3.5),1.10 (3H, s, C18-CH₃), 1.06 (3H, d, J=6.6, C21-CH₃), 0.78 (3H, s,C19-CH₃). ¹³C NMR (140 MHz, CDCl₃): δ=198.3, 162.6, 131.1, 57.9, 54.6,52.9, 52.5, 51.2, 43.2, 40.6, 39.1, 36.9, 35.6, 34.6, 34.1, 33.9, 28.0,23.7, 19.9, 17.7, 17.2 11.9.

I. Epoxidation ofN-((22E)-3,24-dioxo-4,6,22-cholatrien-24-yl)cyclopropyl Sulfonamide toForm N-((6α, 7α,22E)-3,24-dioxo-6,7-epoxy-4,22-choladien-24-yl)cyclopropylsulfonamide

The product was prepared according to the general procedure for MTOcatalysed epoxidation on 1 g scale, isolated in 68% yield (697 mg) as anoff white solid.

δH (400 MHz, CDCl₃); 8.69 (1H, brs, NH), 6.93 (1H, dd, J 15.4, 9.6,C23-CH), 6.12 (1H, s, C4-CH), 5.83 (1H, m, C22-CH), 3.47 (1H, d, J 14.7,C6-CH), 3.36-3.32 (1H, m, C7-CH), 3.00 (1H, dddd, J 12.8, 9.5, 8.1, 4.8,SO₂CH), 2.67-2.40 (2H, m), 2.39-2.27 (1H, m), 2.09-1.64 (7H, m),1.62-1.18 (11H, m), 1.11 (3H, d, J 6.1, C21-CH₃), 1.10 (3H, s, C19-CH₃),0.78 (3H, s, C18-CH₃); δC (100 MHz, CDCl₃); 198.6, 164.0, 162.8, 156.6,131.1, 119.3, 54.6, 54.5, 52.6, 51.2, 43.4, 40.6, 39.8, 39.1, 35.6,34.6, 34.1, 33.9, 31.5, 28.2, 23.7, 19.9, 19.1, 17.2, 12.1, 6.3, 6.3.

J. Epoxidation ofN-((22E)-3,24-dioxo-4,6,22-cholatrien-24-yl)-4-(trifluoromethoxy)benzenesulfonamideto Form N-((6α, 7α,22E)-3,24-dioxo-6,7-epoxy-4,22-choladien-24-yl)-4-(trifluoromethoxy)benzenesulfonamide

The product was prepared according to the general procedure for MTOcatalysed epoxidation on 1 g scale, isolated in 5% yield (50 mg) as acolourless solid.

δH (400 MHz, MeOD); 8.17-8.09 (2H, m, ArH), 7.52-7.46 (2H, m, ArH), 6.82(1H, dd, J 15.4, 8.9, 3.7, C23-CH), 6.07 (1H, s, C4-CH), 5.84 (1H, dd, J15.4, 0.7, C22-CH), 3.49 (1H, d, J 3.8, C6-CH), 3.37-3.33 (1H, m,C7-CH), 2.62 (1H, ddd, J 18.2, 14.6, 5.6, C2-CH_(a)H_(b)), 2.44-2.27(2H, m), 2.08-1.88 (3H, m), 1.85-1.60 (2H, m), 1.60-1.49 (1H, m),1.48-1.17 (9H, m), 1.12 (3H, s, C19-CH₃), 1.07 (3H, d, J 6.6, C21-CH₃),0.80 (3H, s, C18-CH₃); δC (100 MHz, MeOD); 201.0, 166.2, 166.1, 156.5,153.9, 139.8, 131.8, 131.4, 122.0, 121.7 (q, J 256), 120.8, 55.9, 55.7,53.6, 52.8, 44.6, 42.3, 41.0, 40.5, 36.9, 35.9, 35.2, 35.0, 29.2, 24.6,21.0, 19.5, 17.3, 12.4.

K. Epoxidation of(20S)-20-(5-Tosyltetrazol-1-yl)methyl-pregna-4,6-dien-3-one to Form (6α,7α, 20S)-20-(5-Tosyltetrazol-1-yl)methyl-6,7-epoxy-pregna-4-en-3-one

The product was prepared according to the general procedure for MTOcatalysed epoxidation on 300 mg scale, isolated in 33% yield (103 mg) asa colourless solid.

δH (400 MHz, CDCl₃); 8.00-7.94 (2H, m, ArH), 7.47-7.41 (2H, m, ArH),6.10 (1H, s, C4-CH), 4.77 (1H, dd, J 13.4, 3.9, C22-CH_(a)H_(b)), 4.42(1H, dd, J 13.4, 3.9, C22-CH_(a)H_(b)), 3.46 (1H, d, J 3.7, C6-CH),3.37-3.33 (1H, m, C7-CH), 2.61-2.37 (3H, m), 2.48 (3H, s, ArCH₃),2.37-2.24 (1H, m), 2.11-1.80 (3H, m), 1.76-1.61 (2H, m), 1.58-1.17 (8H,m), 1.09 (3H, s, C19-CH₃), 0.85 (3H, d, J 7.0, C21-CH₃), 0.81 (3H, s,C18-CH₃); δC (100 MHz, CDCl₃); 198.2, 162.5, 153.3, 147.5, 134.4, 131.1,130.4, 129.3, 55.1, 54.5, 53.8, 52.5, 51.2, 43.6, 40.6, 39.1, 37.7,35.5, 34.6, 34.1, 33.9, 27.6, 23.8, 21.9, 19.9, 17.2, 16.4, 11.9.

Example 3—Preparation of Compounds of General Formula (XXI) ViaCompounds of General Formula (I) with Malonate Side Chain

The compounds of general formula (II) may be converted to compounds ofgeneral formula (IA) as described above and these compounds may then beconverted to compounds of general formula (IB), (IC), (ID), (IE) and(IF) by the methods described below. A compound of general formula (IF)may be converted to a compound of general (XXI).

The following illustrates the conversion of a compound of generalformula (II) in which —YR⁴ is CH₂OH via compounds of formulae (IA),(IB), (IC), (ID) and (IE) in which —YR⁴ is —CH₂CH[C(O)OMe]₂ to acompound of general formula (XXI) in which R^(4a) is C(O)OH is shown inScheme 4 below.

A. Synthesis of 23-carboxy-3-oxo-4-cholen-24-oic Acid Dimethyl Ester

To a suspension of (20S)-20-bromomethyl-4-pregnen-3-one (15 g, 38.1mmol), tetrabutylammonium bromide (1.2 g, 3.8 mmol) and potassiumcarbonate (26.3 g, 191 mmol) in toluene (150 mL) was addeddimethylmalonate (13.1 mL, 114 mmol) and the reaction mixture wasstirred at 80° C. for 91 h. The reaction mixture was then cooled to roomtemperature and was poured onto water (150 mL). The layers wereseparated and the aqueous phase was extracted with EtOAc (2×100 mL). Thecombined organic phases were washed with 5% aq. sodium chloride (100 mL)and were concentrated under reduced pressure. The residue was purifiedby column chromatography on silica gel (heptane-EtOAc) to give thedesired product (14.8 g, 87%) as a yellow solid. ¹H NMR (700 MHz,CDCl₃): δ=5.72 (1H, s), 3.75 (3H, s), 3.72 (3H, s), 3.48 (1H, dd,J=11.0, 4.0), 2.44-2.36 (2H, m), 2.33 (1H, dt, J=17.0, 3.6), 2.27 (1H,ddd, J=14.6, 4.1, 2.4), 2.18 (1H, ddd, J=13.7, 11.1, 2.5), 2.03-2.00(2H, m), 1.95-1.89 (1H, m), 1.85-1.82 (1H, m), 1.71-1.67 (1H, m),1.64-1.60 (1H, m), 1.54-1.39 (4H, m), 1.37-1.30 (2H, m), 1.19-1.09 (3H,m), 1.18 (3H, s), 1.05-0.99 (2H, m), 0.94-0.90 (1H, m), 0.93 (3H, d,J=6.5), 0.70 (3H, s); ¹³C NMR (176 MHz, CDCl₃): δ=199.6, 171.5, 170.4,170.0, 123.8, 56.3, 55.8, 53.7, 52.6, 52.4, 49.4, 42.5, 39.6, 38.6,35.7, 35.6, 35.1, 34.3, 34.0, 32.9, 32.0, 28.0, 24.1, 21.0, 18.1, 17.4,11.9.

B. Synthesis of 23-carboxy-3-oxo-4,6-choladien-24-oic Acid DimethylEster

23-Carboxy-3-oxo-4-cholen-24-oic acid dimethyl ester (14.5 g, 32.7 mmol)was suspended in toluene (60 mL) and acetic acid (0.19 mL, 3.3 mmol).p-Chloranil (8.8 g, 35.9 mmol) was added and the mixture stirred atreflux for 65 min. The reaction mixture was cooled to room temperatureand filtered. The filter cake was washed with toluene (45 mL) and thefiltrate concentrated under reduced pressure. The residue (21.6 g) wasused without further purification. A small portion was purified bycolumn chromatography on silica gel (heptane-EtOAc) to give the product.¹H NMR (700 MHz, CDCl₃): δ=6.12 (1H, d, J=10.8), 6.08 (1H, dd, J=9.8,2.2), 5.65 (1H, s), 3.74 (3H, s), 3.71 (3H, s), 3.47 (1H, dd, J=11.0,3.9), 2.58 (1H, dd, J=14.3, 5.3), 2.53 (1H, dd, J=14.3, 5.3), 2.44-2.38(1H, m), 2.21-2.15 (2H, m), 2.05-1.92 (3H, m), 1.83-1.77 (1H, m), 1.69(1H, td, J=13.9, 5.2), 1.55-1.34 (5H, m), 1.31-1.11 (5H, m), 1.10 (3H,s), 0.93 (3H, d, J=6.3), 0.73 (3H, s); ¹³C NMR (176 MHz, CDCl₃):δ=199.6, 170.4, 170.0, 163.9, 141.4, 127.8, 123.5, 56.1, 53.4, 52.6,52.4, 50.6, 49.4, 43.5, 39.5, 37.7, 36.0, 35.1, 34.3, 33.9, 33.9, 28.0,23.7, 20.6, 18.1, 16.3, 11.9.

C. Synthesis of (6α, 7α)-6,7-epoxy-3-oxo-4-cholen-23-carboxy-24-oic AcidDimethyl Ester

23-Carboxy-3-oxo-4,6-choladien-24-oic acid dimethyl ester (8.94 g, 19.5mmol) was dissolved in HFIP (35.8 mL) and EtOAc (17.9 mL) and thesolution was cooled to 10° C. MTO (51 mg, 0.195 mmol) and3-methylpyrazole (97 μL, 1.17 mmol) were charged to the solutionfollowed by UHP (2.08 g, 21.4 mmol) in 2 portions over 5 minutes. After2 h further MTO (51 mg, 0.195 mmol) and 3-methylpyrazole (97 μL, 1.17mmol) were charged and the solution stirred for 16 h. Further MTO (51mg, 0.195 mmol), 3 methylpyrazole (97 μL, 1.17 mmol) and UHP (0.38 g,3.90 mmol) were charged and the solution stirred for 2 h. The reactionwas quenched by addition of 5% aq. NaHSO₃ (36 mL) over 5 minutes. Thephases were separated and the organic phase washed with 5% aq. NaHSO₃until a negative test for peroxides was observed. The organic phase waswashed with 5% aq. NaHCO₃ (40 mL) and water (40 mL), then dried oversodium sulfate and was concentrated in vacuo. The residue was purifiedby column chromatography on silica gel to give the desired product (7.07g, 76%) as a white crystalline solid. ¹H NMR (700 MHz, CDCl₃): δ=6.10(1H, s), 5.31 (2H, s), 3.75 (3H, s), 3.73 (3H, s), 3.48 (1H, dd, J=11.1,4.0), 3.45 (1H, d, J=4.0 Hz), 3.34 (1H, d, J=3.6 Hz), 2.55 (1H, ddd,J=18.1, 14.4, 5.6), 2.45 (1H, m), 2.19 (1H, ddd, J=13.6, 11.1, 2.4),2.05-1.85 (5H, m), 1.70 (1H, td, J=13.9, 5.2), 1.53-1.25 (6H, m),1.22-1.17 (2H, m), 1.09 (3H, s), 0.49 (3H, d, J=6.5), 0.72 (3H, s); ¹³CNMR (176 MHz, CDCl₃): δ=198.4, 170.3, 170.0, 162.8, 131.1, 56.0, 54.6,53.4, 52.6, 52.5, 52.4, 51.3, 49.3, 43.1, 40.6, 39.2, 35.5, 35.1, 34.5,34.3, 34.1, 33.8, 28.1, 23.6, 19.9, 18.1, 17.2, 11.8.

D. Synthesis of (6β,7α)-6-ethyl-7-hydroxy-3-oxo-4-cholen-23-carboxy-24-oic Acid DimethylEster

To a solution of 0.5 M ZnCl in THF (15.7 mL, 0.6 eq) in THF (24 mL, 4vol) under argon at −15° C. was added 1 M EtMgBr in TBME (23.6 mL, 1.8eq) dropwise over 20 mins. CuCl (65 mg, 0.05 eq) was added in a singleportion and the suspension stirred for 10 mins. (6α,7α)-6,7-epoxy-3-oxo-4-cholanen-23-carboxy-24-oic acid dimethyl ester (6g) dissolved in THF (24 mL, 4 vol) was added dropwise over 30 mins andthe mixture stirred for 90 mins. Sat. aq. NH₄Cl (15 mL, 2.5 vol) wasadded dropwise and the mixture warmed to ambient temperature. The solidswere removed by filtration and the filter cake washed with EtOAc (2×25mL). The filtrate was washed with sat. aq. NH₄Cl (2×100 mL) and water(2×100 mL). The organic phase was dried over Na₂SO₄, filtered, andconcentrated in vacuo. Purification by column chromatography afforded(6β, 7α)-6-ethyl-7-hydroxy-3-oxo-4-cholanen-23-carboxy-24-oic aciddimethyl ester as a white crystalline solid (55%).

¹H NMR (700 MHz, CDCl₃): δ=5.77 (1H, s), 3.75 (3H, s), 3.74 (1H, s),3.73 (3H, s), 3.48 (1H, dd, J=11.1, 4.0), 2.47 (1H, ddd, J=17.5, 15.0,5.0), 2.37 (1H, m), 2.31 (1H, m), 2.19 (1H, m), 2.05-1.94 (4H, m),1.81-1.41 (11H, m), 1.40-1.34 (2H, m), 1.21 (3H, s), 1.20-1.12 (2H, m),0.93 (3H, d, J=6.4), 0.91 (3H, t, J=7.3), 0.72 (3H, s).

¹³C NMR (176 MHz, CDCl₃): δ=199.1, 170.6, 170.4, 170.0, 128.6, 72.2,56.3, 55.2, 52.6, 52.4, 50.1, 49.4, 44.2, 42.6, 39.1, 38.3, 37.5, 35.6,35.1, 34.4, 34.1, 28.0, 26.3, 23.6, 20.9, 19.7, 18.1, 12.8, 11.8.

E. Synthesis of (5β, 6β,7α)-6-ethyl-7-hydroxy-3-oxo-cholan-23-carboxy-24-oic Acid Dimethyl Ester

A solution of (6β,7α)-6-ethyl-7-hydroxy-3-oxo-4-cholanen-23-carboxy-24-oic acid dimethylester (3.5 g) in DMF (10.5 mL, 3 vol) and MeCN (21 mL, 6 vol) was purgedwith argon×3 and cooled to −15° C. 5% Pd on CaCO₃ was added in oneportion and the flask then purged with hydrogen×3 and stirred for 18 h.The flask was purged with argon×3 times and the suspension filteredthrough a Whatman® GF/B grade filter pad (glass fiber pore size 1 μm)and the cake washed with EtOAc (2×50 mL). The filtrate was washed withwater (2×50 mL) and 5% aq. NaCl (50 mL), dried over Na₂SO₄, filtered andconcentrated in vacuo. Purification by column chromatography gave (5β,6β, 7α)-6-ethyl-7-hydroxy-3-oxo-cholan-23-carboxy-24-oic acid dimethylester (1.77 g, 51%).

¹H NMR (700 MHz, CDCl₃): δ=3.75 (3H, s), 3.73 (3H, s), 3.70 (1H, s),3.48 (1H, dd, J=11.0, 4.0), 3.35 (1H, dd, J=15.5, 13.6), 2.36 (1H, td,J=14.2, 4.8), 2.19 (1H, m), 2.14-2.08 (2H, m), 2.02-1.90 (4H, m), 1.81(1H, dd, J=13.3, 4.5), 1.70-1.62 (2H, m), 1.54-1.34 (11H, m), 1.26-1.11(2H, m), 1.04 (3H, s), 0.95 (3H, d, J=6.4), 0.94 (3H, d, J=7.0), 0.70(3H, s).

¹³C NMR (176 MHz, CDCl₃): δ=213.7, 170.4, 170.1, 72.1, 56.4, 52.6, 52.4,50.2, 49.8, 49.4, 47.0, 46.7, 42.8, 39.5, 37.7, 36.3, 36.0, 35.7, 35.2,34.4, 34.1, 28.1, 27.7, 24.4, 23.8, 20.8, 18.2, 13.9, 11.8.

F. Synthesis of (5β, 6β)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic AcidDimethyl Ester

To a solution of (5β, 6β,7α)-6-ethyl-7-hydroxy-3-oxo-cholan-23-carboxy-24-oic acid dimethyl ester(1.77 g) in DCM (45 mL, 25 vol) under argon was added DMP (1.83 g, 1.2eq) in 4 portions at 5 min intervals. After 30 mins the mixture waspartitioned between EtOAc (50 mL) and 10% aq. Na₂S₂O₃/2% aq. NaHCO₃ andstirred for 1 h. The aqueous phase was extracted with EtOAc (50 mL) andthe combined organic phases washed with 1M aq. NaOH (50 mL). The organicphase was dried over Na₂SO₄, filtered and concentrated in vacuo.Purification by column chromatography gave (5β,6β)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic acid dimethyl ester (1.54g, 87%) as a white crystalline solid.

¹H NMR (700 MHz, CDCl₃): δ=3.75 (3H, s), 3.73 (3H, s), 3.47 (1H, dd,J=10.9, 4.0), 2.42 (1H, t, J=11.4), 2.31-2.17 (5H, m), 2.05 (1H, m),2.01-1.93 (2H, m), 1.89-1.78 (5H, m), 1.67-1.62 (1H, m), 1.58-1.46 (5H,m), 1.39-1.15 (5H, m), 1.14 (3H, s), 0.94 (3H, d, J=6.4), 0.85 (3H, t,J=7.4), 0.71 (3H, s).

¹³C NMR (176 MHz, CDCl₃): δ=214.6, 211.6, 170.4, 170.0, 57.2, 55.5,52.6, 52.4, 50.3, 49.4, 48.5, 47.3, 44.9, 43.6, 43.2, 39.2, 35.8, 35.3,35.1, 34.9, 34.3, 28.1, 24.6, 23.8, 23.5, 21.7, 18.2, 12.6, 12.2.

G. Synthesis of (5β, 6α)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic AcidDimethyl Ester

To (5β, 6β)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic acid dimethylester (1.46 g) in MeOH (36 mL, 25 vol) under argon was added NaOMe (324mg, 2 eq) and the solution stirred at 40° C. for 16 hours. AcOH (5 mL)was added dropwise and the solution stirred for 5 minutes. The solutionwas taken up in EtOAc (100 mL) and washed with 5% aq. NaCl (2×100 mL).The organic phase was dried over Na₂SO₄, filtered and concentrated invacuo. Purification by column chromatography gave (5β,6α)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic acid dimethyl ester (0.45g, 31%).

¹H NMR (700 MHz, CDCl₃): δ=3.75 (3H, s), 3.73 (3H, s), 3.47 (1H, dd,J=11.0, 4.0), 2.74 (1H, dd, J=11.0, 6.6), 2.47 (1H, t, J=11.3),2.29-2.16 (5H, m), 2.09-1.96 (3H, m), 1.89-1.80 (2H, m), 1.72-1.46 (6H,m), 1.39-1.34 (1H, m), 1.33 (3H, s), 1.32-1.23 (2H, m), 1.21-1.13 (2H,m), 1.10-1.07 (1H, m), 0.99-0.95 (1H, m), 0.94 (3H, d, J=6.5), 0.81 (3H,t, J=7.4), 0.68 (3H, s); ¹³C NMR (176 MHz, CDCl₃): δ=212.1, 210.5,170.3, 170.0, 55.3, 52.6, 52.4, 52.3, 52.2, 49.9, 49.34, 48.8, 43.7,42.7, 38.8, 38.3, 36.6, 35.9, 35.4, 35.1, 34.2, 28.2, 24.5, 22.9, 22.2,18.6, 18.2, 12.1, 11.8.

H. Synthesis of (3α, 5β,6α)-6-ethyl-3-hydroxy-7-oxo-cholan-23-carboxy-24-oic Acid Dimethyl Ester

To a suspension of NaBH₄ (27 mg, 1 eq) in IPA (2.3 mL) at −20° C. wasadded a solution of (5β, 6α)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oicacid dimethyl ester (350 mg) in EtOAc (2.3 mL, 6.5 vol) over 10 mins.After 30 mins 0.7M H₂SO₄ (2.5 mL) was added dropwise over 10 mins andthe solution allowed to warm to 18° C. The solution was diluted withEtOAc (50 mL) and the organic phase washed with water (3×50 mL) and 5%aq. NaCl (50 mL). The organic phase was dried over Na₂SO₄, filtered andconcentrated in vacuo. Purification by column chromatography gave (3α,5β, 6α)-6-ethyl-3-hydroxy-7-oxo-cholan-23-carboxy-24-oic acid dimethylester (298 mg, 85%)

¹H NMR (700 MHz, CDCl₃): δ=3.74 (3H, s), 3.72 (3H, s), 3.52 (1H, m),3.47 (1H, dd, J=11.0, 4.0), 2.69 (1H, dd, J=12.8, 5.9), 2.34 (1H, t,J=11.3), 2.21-2.16 (2H, m), 1.99-1.94 (2H, m), 1.85-1.68 (7H, m),1.50-1.43 (4H, m), 1.37-1.23 (5H, m), 1.21 (3H, s), 1.20-1.10 (4H, m),0.92 (3H, d, J=6.5), 0.80 (3H, t, J=7.4), 0.64 (3H, s); ¹³C NMR (176MHz, CDCl₃): δ=212.8, 170.4, 170.0, 71.1, 55.3, 52.6, 52.4, 52.0, 50.7,49.9, 49.4, 49.0, 43.7, 42.7, 39.0, 35.7, 35.1, 34.3, 34.2, 31.8, 29.8,28.3, 24.6, 23.5, 21.8, 18.8, 18.2, 12.0, 12.0.

I. Synthesis of (3α, 5β, 6α,7α)-6-ethyl-3,7-dihydroxy-cholan-23-carboxy-24-oic Acid Dimethyl Ester

To a solution of (3α, 5β,6α)-6-ethyl-3-hydroxy-7-oxo-cholan-23-carboxy-24-oic acid dimethyl ester(200 mg) in THF (20 mL, 100 vol) and water (5 mL, 25 vol) at 0° C. wasadded NaBH₄ (154 mg, 10 eq) in 3 portions. The solution was stirred forone h, allowing to warm to 18° C. MeOH/water (10 mL, 1:1) was addeddropwise and the organic solvent removed in vacuo. To the aqueoussolution was added 2M aq. HCl (20 mL) dropwise. The aqueous solution wasextracted with EtOAc (2×30 mL) and the combined organic phases washedwith 5% aq.NaHCO₃ (30 mL) and water (30 mL). The organic phase was driedover Na₂SO₄, filtered and concentrated in vacuo. Purification by columnchromatography gave (3α, 5β, 6α,7α)-6-ethyl-3,7-dihydroxy-cholan-23-carboxy-24-oic acid dimethyl ester(90 mg, 45%).

¹H NMR (700 MHz, CDCl₃): δ=3.75 (3H, s), 3.72 (3H, s), 3.48 (1H, dd,J=11.0, 4.0), 3.69 (1H, bs), 3.40 (1H, m), 2.18 (1H, m), 1.97-1.93 (2H,m), 1.85-1.75 (4H, m), 1.73-1.57 (4H, m), 1.51-1.11 (18H, m), 1.00 (1H,td, J=14.3, 3.4), 0.93 (3H, d, J=6.5), 0.90 (3H, t, J=7.3), 0.64 (3H,s); ¹³C NMR (176 MHz, CDCl₃): δ=170.5, 170.1, 72.3, 70.9, 56.3, 52.6,52.4, 50.5, 49.4, 45.2, 42.8, 41.2, 40.0, 39.6, 35.6, 35.5, 35.2, 34.4,34.0, 33.2, 30.6, 28.2, 23.7, 23.2, 22.2, 20.7, 18.2, 11.8, 11.7.

J. Synthesis of (3α, 5β, 6α,7α)-6-ethyl-3,7-dihydroxy-cholan-23-carboxy-24-oic Acid

To a solution of (3α, 5β, 6α,7α)-6-ethyl-3,7-dihydroxy-cholan-23-carboxy-24-oic acid dimethyl ester(70 mg) in IPA (2 mL, 28 vol) was added 0.5 M aqueous NaOH (2 mL, 28vol) and the mixture stirred at 60° C. for 2 h. The organic solvent wasremoved in vacuo and the aqueous solution adjusted to pH1 with 2M aq.H₂SO₄. EtOAc (20 mL) was added and the mixture stirred for 5 mins. Theaqueous phase was re-extracted with EtOAc (10 mL). The combined organicphases were washed with 5% aq.NaCl (2×10 mL), dried over Na₂SO₄,filtered and concentrated in vacuo to give (3α, 5β, 6α,7α)-6-ethyl-3,7-dihydroxy-cholan-23-carboxy-24-oic acid as a white solid(54 mg, 81%).

¹H NMR (700 MHz, d-6 Acetone): δ=3.58 (1H, bs), 3.32 (1H, dd, J=11.1,3.6), 3.18 (1H, m), 2.03 (1H, m), 1.90-1.62 (6H, m), 1.57 (1H, m),1.48-1.31 (8H, m), 1.28-1.13 (6H, m), 1.11-1.05 (3H, m), 0.98 (3H, m),0.87 (3H, d, J=6.1), 0.85 (1H, m) 0.79 (3H, s), 0.75 (3H, t, J=7.3),0.74 (3H, s); ¹³C NMR (176 MHz, d-6 Acetone): δ=171.7, 171.3, 72.5,70.4, 57.5, 51.4, 46.7, 43.4, 42.6, 41.3, 40.7, 36.7, 36.3, 36.2, 35.3,34.6, 34.0, 31.5, 30.6, 29.0, 24.3, 23.7, 23.2, 21.6, 18.7, 12.3, 12.1.

K. Synthesis of (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-24-oicAcid

(3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-23-carboxy-24-oic acid (25mg) was taken up in xylene (1.25 mL, 50 vol) and pyridine (250 μL, 10vol) and the solution heated to reflux for 90 mins. The cooled solutionwas diluted with EtOAc (20 mL) and washed with 1M aq. HCl (3×10 mL). Theorganic phase was washed with water (3×10 mL), 5% aq. NaCl (10 mL),dried over Na₂SO₄, filtered and concentrated. Purification by columnchromatography gave (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-24-oicacid as a white solid (19 mg, 82%).

¹H and ¹³C NMR were consistent with an authentic sample of (3α, 5β, 6α,7α)-6-ethyl-3,7-dihydroxy-cholan-24-oic acid.

L. Synthesis of (5β, 6α)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic Acid

To a solution of (5β, 6β)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oicacid dimethyl ester (100 mg) in IPA (1 mL, 10 vol) was added 0.5 Maqueous NaOH (1 mL, 10 vol) and the mixture stirred at 60° C. for 2 h.The organic solvent was removed in vacuo and the aqueous solutionadjusted to pH1 with 2M aqueous H₂SO₄. EtOAc (10 mL) was added and themixture stirred 5 mins. The aqueous phase was re-extracted with EtOAc(10 mL). The combined organic phases were washed with 5% aq. NaCl (2×10mL), dried over Na₂SO₄, filtered and concentrated in vacuo to give (5β,6α)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic acid (100 mg, quant.) asa white solid.

¹H NMR (500 MHz, CDCl₃): δ=3.51 (1H, m), 2.76 (1H, m), 2.49 (1H, t,J=11.1), 2.34-1.80 (14H, m), 1.71-1.43 (7H, m), 1.33 (3H, s), 1.23-1.04(3H, m), 0.98 (3H, d, J=6.1), 0.94 (1H, m), 0.80 (3H, d, J=7.3), 0.69(3H, s).

M. Synthesis of (5β, 6α)-6-ethyl-3,7-dioxo-cholan-24-oic Acid

(5β, 6α)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oic acid (80 mg) wastaken up in xylene (4 mL, 50 vol) and pyridine (800 μL, 10 vol) and thesolution heated to reflux for 90 mins. The cooled solution was dilutedwith EtOAc (25 mL) and washed with 1M aq.HCl (3×10 mL). The organicphase was washed with water (3×10 mL), 5% aq. NaCl (10 mL), dried overNa₂SO₄ and filtered. Purification by column chromatography gave (5β,6α)-6-ethyl-3,7-dioxo-cholan-24-oic acid as a white solid (60 mg, 83%).

¹H and ¹³C NMR were consistent with an authentic sample of the targetcompound, prepared as described in Example 16 of WO 2016/079520.

Although the compound of general formula (XXI) was prepared from acompound of general formula (IF) by conversion of the malonate sidechain of the compound of general formula (IF) to a carboxylic acidgroup, a person of skill in the art will appreciate that the conversionof the malonate to carboxylic acid could take place at an earlier stageof the synthesis as described in steps M and N above, and that thecarboxylic acid group could, if necessary, be protected, for example asan ester.

Example 4—Preparation of an Analogue of a Compound of General Formula(I) and a Compound of General Formula (XXI) Via Compounds of GeneralFormula (I) with Nitrile Side Chain (Including Side Chain Extension)

Scheme 5 shows the conversion of a compound of general formula (II) inwhich —YR⁴ is CH₂OH conversion to a compound of general formula (II) inwhich —YR⁴ is —CH₂CH₂—CN and subsequently to a compound of generalformula (XXI) in which —YR^(4a) is CH₂CH₂C(O)OH. The reaction proceedsvia compounds of general formulae (IA), (IB), (IC) and (IE) in which—YR⁴ is —CH₂CH₂—CN. The compound of general formula (IE) is thenconverted to a 3-OH analogue and the side chain is then converted to—CH₂CH₂—C(O)OH.

A. Synthesis of (20S)-20-bromomethyl-3,3-ethylenedioxy-4-pregnene and(20S)-20-bromomethyl-3,3-ethylenedioxy-5-pregnene

To a solution of (20S)-20-bromomethyl-4-pregnene-3-one (1.00 g, 2.59mmol) and ethylene glycol (2.0 mL, 36.25 mmol) in toluene (30 mL) wasadded pTSA.H₂O (9.86 mg, 0.05 mmol) and the mixture was heated to refluxusing a Dean Stark apparatus for 5 h. The reaction mixture was allowedto cool to room temperature before being poured onto 5% aq. NaHCO₃ (30mL). The layers were separated and the aqueous layer was extracted withCH₂Cl₂ (2×30 mL). The combined organics were dried over sodium sulfateand were concentrated under reduced pressure. The residue was used inthe next step without purification. A sample was purified by columnchromatography (heptane/EtOAc) to give a mixture of(20S)-20-bromomethyl-3,3-ethylenedioxy-4-pregnene and(20S)-20-bromomethyl-3,3-ethylenedioxy-5-pregnene in 68% yield (theratio of Δ⁵:Δ⁴ was approximately 3.6:1). δH (700 MHz, CDCl₃); 5.35(0.8H, dt, J=4.4, 2.2), 5.23 (0.2H, s), 4.02-3.96 (4H, m, CH₂O), 3.51(0.8H, dd, J 9.7, 2.7), 3.51-3.49 (0.2H, m), 3.34 (0.8H, dd, J 9.7,6.0), 3.33 (0.2H, dd, J 9.7, 6.1), 2.56 (0.8H, dq, J 14.1, 2.9), 2.20(0.2H, td, J 13.9, 4.9, 1.8), 2.12 (0.8H, dd, J 14.2, 2.9), 2.05 (0.2H,ddd, J 14.0, 4.2, 2.4), 1.99-1.93 (2H, m), 1.91-1.83 (1H, m), 1.81-1.75(2H, m), 1.74-1.62 (4H, m), 1.60 (0.8H, s), 1.561.51 (1H, m), 1.50-1.41(2H, m), 1.37-1.25 (3H, m), 1.21 (1H, td, J 6.5, 4.2), 1.17-1.04 (3H,m), 1.09 (3H, d, J 6.4), 1.03 (3H, s), 1.01-0.84 (0.8H, m), 0.71 (2.4H,s), 0.70 (0.6H, s); δC (176 MHz, CDCl₃); 151.6, 140.2, 122.1, 119.65,109.5, 106.2, 64.6, 64.5, 64.2, 64.2, 56.4, 55.7, 53.8, 53.7, 53.7,49.6, 43.6, 43.5, 42.5, 42.4, 41.8, 39.5, 39.5, 37.9, 37.8, 37.4, 36.6,36.3, 35.8, 34.9, 32.4, 32.1, 31.9, 31.9, 31.7, 31.1, 30.0, 27.6, 27.6,24.2, 24.1, 21.0, 18.9, 18.7, 18.6, 17.6, 12.3, 12.2.

B. Synthesis of 3,3-ethylenedioxy-4-choleno-24-nitrile and3,3-Ethylenedioxy-5-choleno-24-nitrile

Procedure A

A solution containing MeCN (26.0 mg, 0.63 mmol) in THF (1.85 mL) wascooled to −78° C. under argon and nBuLi (0.32 mL, 2 M in cyclohexane,0.63 mmol) was charged dropwise over 2 min. To this mixture, a solutioncontaining (20S)-20-bromomethyl-3,3-ethylenedioxy-4-pregnene and(20S)-20-bromomethyl-3,3-ethylenedioxy-5-pregnene (185 mg, 0.423 mmol)in THF (2.15 mL) was charged dropwise over 30 min. The reaction mixturewas allowed to warm to 0° C. over 4 h, cooled to −78° C. and quenchedwith 10% aq. NH₄Cl (3 mL). The reaction mixture was diluted with EtOAc(20 mL) and 10% aq. NH₄Cl (20 mL) and the organic phase was separated.The aqueous phase was extracted with EtOAc (20 mL), and the combinedorganic phases were washed with 5% aq. NaCl (20 mL), dried over sodiumsulfate and concentrated under reduced pressure. The residue waspurified by column chromatography on silica gel using heptane:EtOAc(5:1) as the eluent. A fraction containing3,3-ethylenedioxy-4-choleno-24-nitrile and3,3-ethylenedioxy-5-choleno-24-nitrile was obtained in 49% yield (theratio of Δ⁵:Δ⁴ was approximately 7:1). δH (700 MHz, CDCl₃); 5.35 (0.9H,dt, J 4.5, 2.2), 5.2 (0.1H, brs), 4.02-3.86 (4H, m), 2.56 (0.9H, dq, J14.2, 2.9), 2.39-2.34 (0.1H, m), 2.34 (0.9H, ddd, J 16.9, 8.6, 5.1),2.27 (0.9H, dt, J 16.8, 8.4), 2.27 (0.1H, dt, J 16.8, 8.4), 2.20 (0.1H,td, J 13.9, 5.0, 1.8), 2.12 (0.9H, dd, J 14.2, 3.0), 2.05 (0.1H, ddd, J13.8, 4.4, 2.2), 2.01-1.95 (2H, m), 1.87-1.75 (4H, m), 1.73-1.70 (0.3H,m), 1.69-1.59 (3.4H, m), 1.58-1.52 (2H, m), 1.50-1.43 (2H, m), 1.39-1.25(4.6H, m), 1.18 (1H, td, J 6.5, 4.2), 1.14-0.99 (4H, m), 1.03 (3H, s),0.96 (2.7H, d, J 6.6), 0.94 (0.3H, d, J 6.7), 0.88 (0.9H, t, J 14.3),0.70 (2.7H, s), 0.70 (0.3H, s); δC (176 MHz, CDCl₃); 151.6, 140.1,122.1, 120.2, 119.6, 109.5, 106.2, 64.6, 64.4, 64.2, 56.7, 56.0, 55.5,55.5, 53.8, 49.6, 42.6, 42.5, 41.8, 39.8, 39.7, 37.4, 36.6, 36.3, 35.7,35.2, 35.2, 34.9, 32.4, 32.1, 31.9, 31.9, 31.7, 31.6, 31.5, 31.1, 30.0,29.7, 28.1, 28.1, 24.2, 24.1, 22.7, 21.0, 18.9, 17.9, 17.9, 17.6, 14.3,14.2, 14.1, 12.0, 11.9.

Procedure B

A solution of MeCN (2.06 mL, 39.43 mmol) in THF (34 mL) was chargeddropwise over 1.2 h to a solution of nBuLi (19.72 mL, 2 M incyclohexane, 39.43 mmol) in THF (69 mL) at −60° C. under argon. To theresulting white suspension, a solution containing(20S)-20-bromomethyl-3,3-ethylenedioxy-4-pregnene and(20S)-20-bromomethyl-3,3-ethylenedioxy-5-pregnene (6.9 g, 15.77 mmol) inTHF (69 mL) was charged dropwise over 1.2 h. The thick suspension thatformed was warmed to 0° C. over 15 min and water (69 mL) was chargeddropwise. The layers were separated and the aqueous phase was extractedwith EtOAc (2×100 mL). The combined organic phases were washed with 5%aq. NaCl (2×100 mL) and concentrated under reduced pressure. The residuewas purified by column chromatography on silica gel using a gradient ofEtOAc in heptane as the eluent. A fraction containing3,3-ethylenedioxy-4-choleno-24-nitrile and3,3-ethylenedioxy-5-choleno-24-nitrile was obtained which also containedthe product from double-alkylation of MeCN (mass 3.88 g).

C. Synthesis of 3-oxo-4-choleno-24-nitrile

To a solution of 3,3-ethylenedioxy-4-choleno-24-nitrile and3,3-ethylenedioxy-5-choleno-24-nitrile (3.75 g, 9.43 mmol) in EtOH (75mL) was added a solution of H₂SO₄ (1 mL, conc, 18.86 mmol) in water (7.5mL). The reaction mixture was heated at reflux for 30 min and cooled toroom temperature. A white solid was removed by filtration and thefilter-cake was washed with EtOH (2×20 mL). Pyridine (3 mL) was added tothe combined wash and filtrate and the mixture was concentrated underreduced pressure. The residue was dissolved in EtOAc (100 mL), washedwith 1 M aq. H₂SO₄ (100 mL), 5% aq. NaHCO₃ (100 mL), 5% aq. NaCl (2×100mL), dried over sodium sulfate and was concentrated under reducedpressure to give the desired product (2.36 g). ¹H NMR (700 MHz, CDCl₃):δ=5.72 (1H, s, C4-CH), 2.45-2.25 (6H, m), 2.04-2.00 (2H, m), 1.89-1.82(3H, m), 1.69 (1H, td, J 7.0, 4.6), 1.67-1.62 (1H, m), 1.59-1.51 (3H,m), 1.44 (1H, qd, J 13.1, 4.0), 1.39-1.25 (3H, m), 1.20-1.10 (3H, m),1.18 (3H, s), 1.05-0.99 (2H, m), 0.96 (3H, d, J 6.6), 0.95-0.91 (1H, m),0.73 (3H, s); ¹³C NMR (176 MHz, CDCl₃): δ=199.6 (C═O), 171.4 (C═CH),123.8 (C═CH), 120.2 (CN), 55.8, 55.5, 53.7, 42.6, 39.6, 38.6, 35.7,35.6, 35.1, 34.0, 32.9, 32.0, 31.5, 28.1, 24.1, 21.0, 17.9, 17.4, 14.3,12.0

D. Synthesis of 3-oxo-4,6-choladieno-24-nitrile

To a solution of 3-oxo-4-choleno-24-nitrile (2.25 g, 0.64 mmol) intoluene (2.25 mL) and AcOH (6.75 mL) was added chloranil (1.72 g, 0.70mmol). The mixture was heated at 100° C. for 45 min and was then allowto cool to room temperature. The mixture was filtered, washing withAcOH:toluene (3:1, 20 mL) and the combined filtrates were concentratedunder reduced pressure. The residue was concentrated from toluene (3×40mL) and acetone (3×40 mL) and was then dissolved in acetone (6.75 mL).The solution was charged to an aqueous solution of NaOH (22.5 mL, 3%w/v) and the sticky solid that formed was collected by filtration andwashed with water:acetone (2×20 mL, 2:1). The solid was purified bychromatography on silica gel using a gradient of EtOAc in heptane as theeluent to give the desired product as a yellow solid (1.33 g, 59%yield). ¹H NMR (700 MHz, CDCl₃): δ=6.13 (1H, d, J 11.0), 6.10 (1H, dd, J9.8, 2.3), 5.67 (1H, s), 2.57 (1H, ddd, J 17.9, 14.5, 5.4), 2.45-2.41(1H, m), 2.39 (1H, ddd, J 17.0, 8.3, 5.1), 2.29 (1H, dt, J 16.8, 8.4),2.20 (1H, t, J 10.6), 2.05 (1H, dt, J 12.9, 3.4), 2.00 (1H, ddd, J 13.2,5.3, 2.0), 1.95-1.89 (1H, m), 1.88-1.80 (2H, m), 1.71 (1H, td, J 9.7,1.3), 1.62-1.54 (2H, m), 1.44 (1H, qd, J 9.7, 1.3), 1.41-1.34 (2H, m),1.30 (1H, ddd, J 24.0, 11.7, 5.8), 1.25-1.19 (3H, m), 1.17 (1H, q, J9.5), 1.11 (3H, s), 0.97 (3H, d, J 6.7), 0.78 (3H, s); ¹³C NMR (176 MHz,CDCl₃): δ=199.6, 163.8, 141.1, 127.9, 123.6, 120.1, 55.4, 53.4, 50.6,43.6, 39.5, 37.7, 36.0, 35.2, 34.0, 33.9, 31.4, 28.1, 23.7, 20.6, 17.9,16.3, 14.4, 11.9

E. Synthesis of (6α, 7α)-6,7-epoxy-3-oxo-4-choleno-24-nitrile

A solution of 3-oxo-4,6-choladieno-24-nitrile (1.25 g, 3.56 mmol) inEtOAc (2.5 mL) and HFIP (5 mL) under argon was cooled to 10° C. MTO (8.9mg, 0.036 mmol), 3-methylpyrazole (0.017 mL, 0.213 mmol) and UHP (0.37g, 3.91 mmol) were charged and the mixture was stirred for 2 h. Furtherportions of MTO (8.9 mg, 0.036 mmol), 3-methylpyrazole (0.017 mL, 0.213mmol) and UHP (67 mg, 0.71 mmol) were charged and the mixture wasstirred overnight at 10° C. The reaction was quenched by addition of 5%aq. NaHSO₃ (15 mL) was charged and the mixture was extracted with EtOAc(20 mL). The aqueous phase was separated and extracted with EtOAc (20mL). The combined organic phases were washed with 5% aq. NaCl (20 mL)and were concentrated under reduced pressure. The residue was purifiedby column chromatography on silica gel using a gradient of EtOAc inheptane as the eluent to give the desired product (0.92 g, 70%). ¹H NMR(700 MHz, CDCl₃): δ=6.11 (1H, s), 3.46 (1H, d, J 3.7), 3.34 (1H, d, J3.6), 2.55 (1H, ddd, J 18.1, 14.3, 5.5), 2.47-2.44 (1H, m), 2.41-2.37(1H, ddd, J 16.9, 8.3, 5.0), 2.30 (1H, dt, J 16.8, 8.4), 2.01 (1H, dt, J12.9, 3.3), 1.98-1.83 (5H, m), 1.71 (1H, td, J 6.9, 5.2), 1.61-1.56 (1H,m), 1.52 (1H, dq, J 12.7, 3.6), 1.46 (1H, ddd, J 12.4, 11.4, 7.0),1.41-1.26 (5H, m), 1.22-1.17 (2H, m), 1.10 (3H, s), 0.97 (3H, d, J 6.6),0.76 (3H, s); ¹³C NMR (176 MHz, CDCl₃): δ=198.3, 162.6, 131.1, 120.1,55.3, 54.6, 52.6, 51.3, 43.2, 50.6, 39.3, 35.6, 35.1, 34.6, 34.1, 33.9,31.4, 28.2, 23.6, 19.9, 17.8, 17.2, 14.4, 11.8

F. Synthesis of (6β, 7α)-6-ethyl-7-hydroxy-3-oxo-4-choleno-24-nitrile

A solution of 0.5 M ZnCl₂ in THF (4.65 mL, 2.33 mmol) was cooled to −15°C. and a solution of 1M EtMgBr in TBME (4.65 mL, 4.65 mmol) was chargeddropwise over 1 h. A solution of (6α,7α)-6,7-epoxy-3-oxo-4-choleno-24-nitrile (0.95 g, 2.58 mmol) in THF(4.75 mL) was charged to the resulting mixture over 30 mins. Furtherportions of 1 M EtMgBr in TBME (4.65 mL, 4.65 mmol and 2.33 mL, 2.33mmol) were charged after 15 and 20 mins respectively. The reactionmixture was quenched by addition of a sat. aq. NH₄Cl (2 mL), filteredand the filter-cake washed with TBME (20 mL). The filtrate was washedwith sat. aq. NH₄Cl (3×20 mL), 5% w/v aq. NaCl (2×20 mL) andconcentrated. The residue was purified by column chromatography usingEtOAc in heptane to give (6β,7α)-6-ethyl-7-hydroxy-3-oxo-4-choleno-24-nitrile in 37% yield. ¹H NMR(700 MHz, CDCl₃): δ=5.78 (1H, s), 3.73 (1H, s), 2.48 (1H, ddd, J=17.5,15.1, 4.9), 2.40-2.36 (2H, m), 2.32-2.26 (2H, m), 2.04-2.00 (2H, m),1.94-1.89 (1H, m), 1.87-1.83 (1H, m), 1.81-1.73 (2H, m), 1.70 (1H, td,J=11.3, 2.1), 1.64-1.42 (8H, m), 1.40-1.33 (2H, m), 1.27-1.13 (3H, m),1.22 (3H, s), 0.97 (3H, d, J=6.6), 0.92 (3H, t, J=7.4), 0.76 (3H, s);¹³C NMR (176 MHz, CDCl₃): δ=199.1, 170.4, 128.7, 120.1, 72.2, 55.5,55.3, 50.1, 44.3, 42.6, 39.2, 38.3, 37.5, 35.6, 35.2, 34.1, 31.5, 28.0,26.3, 23.6, 20.9, 19.7, 17.8, 14.3, 12.8, 11.9

G. Synthesis of (5β, 6β, 7α)-6-ethyl-7-hydroxy-3-oxo-cholano-24-nitrile

To a solution of (6β, 7α)-6-ethyl-7-hydroxy-3-oxo-4-choleno-24-nitrile(350 mg, 0.88 mmol) in DMF (2.1 mL) under argon, was charged Pd/C (83mg, 10% Pd, 45% in H₂O). The reaction vessel was purged with H₂ andstirred under H₂ overnight. The Pd/C was removed by filtration through aPTFE syringe filter and the filter rinsed with TBME (6×2 mL). Thefiltrate was washed with 5% w/vaq. NaCl (2×10 mL). The aqueous phase wasextracted with TBME and the combined organic phases were concentrated toan oily residue. Purification of the residue by column chromatographyusing EtOAc in heptane to afford (5β, 6β,7α)-6-ethyl-7-hydroxy-3-oxo-cholano-24-nitrile in 74% yield. ¹H NMR (700MHz, CDCl₃): δ=3.71 (1H, br, s), 3.34 (1H, dd, J=15.5, 13.4), 2.41-2.33(2H, m), 2.30 (1H, dt, J=16.8, 8.4), 2.15-2.09 (2H, m), 2.02 (1H, dt,J=12.8, 3.5), 1.98 (1H, dd, J=11.9, 4.6), 1.94-1.89 (2H, m), 1.88-1.83(1H, m), 1.82 (1H, dd, J=13.4, 4.6), 1.71-1.67 (1H, m), 1.65 (1H, td,J=5.6, 2.8), 1.60-1.14 (17H, m), 1.05 (3H, s), 0.98 (3H, d), 0.94 (3H,t, J=7.2), 0.88 (1H, t, J=7.1), 0.73 (3H, s); ¹³C NMR (176 MHz, CDCl₃):δ=213.5, 120.2, 72.1, 55.6, 50.2, 49.9, 47.0, 46.7, 42.8, 39.5, 37.7,36.3, 36.0, 35.7, 35.2, 34.2, 31.5, 28.1, 27.7, 24.4, 23.8, 20.8, 17.9,14.3, 13.9, 11.8

H. Synthesis of (5β, 6β)-3,7-dioxo-6-ethyl-cholano-24-nitrile

To a solution of (5β, 6β, 7α)-6-ethyl-7-hydroxy-3-oxo-cholano-24-nitrile(245 mg, 0.61 mmol) in DCM (6.13 mL) under argon, was added DMP (312 mg,0.74 mmol) in two portions, 5 min apart. The resulting pink suspensionwas stirred for 30 mins and quenched by addition of 10% w/v aq.Na₂S₂O₃:2% w/v aq. NaHCO₃ (5 mL). The aqueous phase was extracted withTBME (3×20 mL) and the combined organic phases were washed with 5% w/vaq.NaCl (20 mL) and concentrated. The residue was purified by columnchromatography using EtOAc in heptane to afford (5β,6β)-3,7-dioxo-6-ethyl-cholano-24-nitrile in 88% yield. ¹H NMR (700 MHz,CDCl₃): δ=2.45 (1H, t, J=11.4), 2.38 (1H, ddd, J=16.9, 8.2, 5.1),2.31-2.20 (5H, m), 2.06 (1H, dt, J=12.9, 3.4), 1.99 (1H, quintet,J=4.7), 1.92-1.78 (7H, m), 1.65 (1H, ddd, J=14.4, 9.9, 4.6), 1.60-1.53(4H, m), 1.52-1.47 (1H, m), 1.40-1.29 (2H, m), 1.25-1.14 (3H, m), 1.16(3H, s), 0.98 (3H, d, J=6.6), 0.84 (3H, t, J=7.4), 0.74 (3H, s); ¹³C NMR(176 MHz, CDCl₃): δ=214.5, 211.5, 120.1, 57.4, 54.8, 50.1, 48.6, 47.2,44.8, 43.7, 43.2, 39.1, 35.8, 35.3, 35.1, 35.0, 31.4, 28.2, 24.6, 23.9,23.6, 21.7, 18.0, 14.3, 12.7, 12.3

I. Synthesis of (3α, 5β, 6β)-6-ethyl-3-hydroxy-7-oxo-cholano-24-nitrile

To a suspension of NaBH₄ (19 mg, 0.50 mmol) in IPA (0.8 mL) cooled to−20° C. was charged a solution of (5β,6β)-3,7-dioxo-6-ethyl-cholano-24-nitrile (200 mg, 0.50 mmol) in EtOAc(1.3 mL) dropwise over 13 mins. A solution of 0.5 M H₂SO₄ (0.5 mL) inwater (0.8 mL) was charged slowly and the reaction mixture was stirredover 15 min and diluted with water (10 mL). The mixture was extractedwith EtOAc (3×10 mL) and the combined organic phases were washed with 5%w/v aq. NaCl (3×10 mL) and concentrated. The residue was purified bycolumn chromatography using EtOAc in heptane to afford (3α, 5β,6β)-6-ethyl-3-hydroxy-7-oxo-cholano-24-nitrile in 73% yield. ¹H NMR (700MHz, CDCl₃): δ=3.59-3.55 (1H, m), 2.57 (1H, dd, J, 11.9, 10.8), 2.38(1H, ddd, J 16.9, 8.4, 5.0), 2.28 (1H, dt, J 16.8, 8.4), 2.20-2.16 (1H,m), 2.00-1.94 (2H, m), 1.93-1.83 (3H, m), 1.81-1.72 (3H, m), 1.70-1.64(3H), 1.57-0.53 (1H, m), 1.52-1.43 (4H, m), 1.39-1.34 (1H, m), 1.32-1.25(2H, m), 1.22 (3H, s), 1.19-1.11 (4H, m), 0.96 (3H, d, J 6.6), 0.95-0.90(1H, m), 0.85 (3H, t, J 7.3), 0.69 (3H, s); ¹³C NMR (176 MHz, CDCl₃):δ=215.3, 120.1, 70.6, 62.1, 54.6, 49.6, 48.7, 45.5, 42.9, 42.6, 39.8,38.8, 35.6, 35.4, 35.0, 31.5, 29.6, 28.2, 26.6, 26.0, 24.9, 21.4, 18.0,14.3, 13.1, 12.2

J. Synthesis of (3α, 5β, 6α)-6-ethyl-3-hydroxy-7-oxo-cholan-24-oic Acid

A mixture of (3α, 5β, 6β)-6-ethyl-3-hydroxy-7-oxo-cholano-24-nitrile(130 mg, 0.33 mmol) in MeOH (6 mL), water (6 mL) and KOH (1.8 g, 32.14mmol) was heated at reflux over 7 h, stirred at ambient for 16 h, thenheated at reflux for a further 4 h. The reaction mixture was cooled toambient temperature and acidified to pH1 with 6M HCl. The mixture wasextracted with EtOAc (3×20 mL) and the combined organic phases werewashed with 5% w/v aq. NaCl (20 mL) and concentrated to give crude (3α,5β, 6α)-6-ethyl-3-hydroxy-7-oxo-cholan-24-oic acid in 82% yield. ¹H and¹³C NMR matched those of an authentic sample.

The product of step J, can be converted to a compound of general formula(XXI) in which R^(4′) is C(O)OH by reduction, for example using sodiumborohydride.

As a person of skill in the art would appreciate, the synthetic routeshown in Scheme 5 could be adapted by conversion of the nitrile group toa carboxylic acid at an earlier stage followed, if necessary, byprotection of the carboxylic acid group, for example as an ester.

Example 5—Preparation of an Analogue of a Compound of General Formula(I) and a Compound of General Formula (XXI) Via Compounds of GeneralFormula (I) with Nitrile Side Chain (not Including Side Chain Extension)

Scheme 6 shows an alternative route in which the side chain is notextended.

A. Synthesis of (20R)-cyanomethyl-4-pregnen-3-one

To a suspension of (20S)-20-bromomethyl-4-pregnen-3-one (15 g, 38.1mmol) in DMF (225 mL) was added potassium cyanide (7.5 g, 114 mmol). Thesuspension was stirred at 80° C. for 41 h before cooling to roomtemperature. EtOAc (250 mL) and water (500 mL) were added and the layerswere separated. The aqueous layer was extracted with EtOAc (2×250 mL)and the combined organic phases were washed with 5% aq. NaCl (250 mL)and were concentrated under reduced pressure. The residue was purifiedby column chromatography on silica gel (heptane/EtOAc) to afford thedesired product (9.7 g, 75%) as a white solid. δH (700 MHz, CDCl₃); 5.73(1H, s, C4-CH), 2.45-2.32 (4H, m), 2.27 (1H, ddd, J=14.6, 4.2, 2.7),2.24 (1H, dd, J=16.8, 7.1), 2.04-1.99 (2H, m), 1.89-1.78 (3H, m),1.72-1.65 (2H, m), 1.57-1.51 (2H, m), 1.43 (1H, qd, J=13.2, 4.0),1.31-1.16 (4H, m), 1.18 (3H, s), 1.17 (3H, d, J=6.7), 1.11-1.01 (2H, m),0.94 (1H, ddd, J=12.3, 10.7, 4.1), 0.74 (3H, s); δC (176 MHz, CDCl₃);199.5, 171.2, 123.9, 118.9, 55.7, 54.7, 53.6, 42.5, 39.2, 38.5, 35.7,35.6, 34.0, 33.6, 32.8, 31.9, 28.0, 24.8, 24.1, 20.9, 19.3, 17.4, 12.1.

B. Synthesis of (20R)-cyanomethyl-4,6-pregnadien-3-one

To a suspension of (20R)-cyanomethyl-4-pregnen-3-one (9.1 g, 26.8 mmol)in toluene (36 mL) and acetic acid (0.15 mL) was added p-chloranil (7.2g, 39.5 mmol). The mixture was heated at reflux for 90 minutes beforeallowing to cool to room temperature. The suspension was filtered,washing with toluene (25 mL). The filtrate was concentrated underreduced pressure and the residue was purified by column chromatographyon silica gel (heptane/EtOAc). The material was then dissolved inacetone (35 mL) and methanol (23 mL) and 0.5 M aq. NaOH (200 mL) wasadded dropwise. Water (100 mL) was added and the resulting solid wasfiltered, washing with water (2×50 mL) and 2:1 acetone:water (2×20 mL).The solid was dried in vacuo to afford the desired product (5.4 g, 60%)as a pale brown solid. δH (700 MHz, CDCl₃); 6.11 (2H, s), 5.67 (1H, s),2.57 (1H, ddd, J=18.0, 14.4, 5.4), 2.45-2.42 (1H, m), 2.37 (1H, dd,J=16.7, 3.7), 2.25 (1H, dd, J=16.7, 7.2), 2.01 (1H, t, J=10.4), 2.03(1H, dt, J=12.8, 3.3), 2.00 (1H, ddd, J=13.2, 5.4, 2.1), 1.96-1.91 (1H,m), 1.88-1.81 (1H, m), 1.74-1.70 (1H, m), 1.58 (1H, dq, J=13.4, 3.6),1.44 (1H, qd, J=4.4, 3.9), 1.36-1.20 (7H, m), 1.18 (3H, d, J=6.7), 1.11(3H, s), 0.79 (3H, s); δC (176 MHz, CDCl₃); 199.6, 163.67, 140.8, 128.1,123.7, 118.8, 54.6, 53.2, 50.5, 43.5, 39.1, 37.6, 36.0, 33.9, 33.9,33.5, 28.0, 24.8, 23.6, 20.6, 19.3, 16.3, 12.0.

C. Epoxidation of (20R)-20-(1-cyanomethyl)-pregna-4,6-dien-3-one to Form(6α, 7α, 20R)-20-(1-cyanomethyl)-6,7-epoxy-pregn-4-en-3-one

(20R)-Cyanomethyl-4,6-pregnadien-3-one (5.1 g, 15.1 mmol) was dissolvedin HFIP (20 mL) and EtOAc (10 mL) and was cooled to 10° C. MTO (38 mg, 1mol %), 3-methylpyrazole (73 μL, 6 mol %) and UHP (1.6 g, 16.6 mmol)were added and the mixture stirred at 10° C. After 4 h, MTO (38 mg, 1mol %), 3-methylpyrazole (73 μL, 6 mol %) and UHP (0.28 g, 3.0 mmol)were added and the mixture stirred at 10° C. After a further 17 h, MTO(38 mg, 1 mol %), 3-methylpyrazole (73 μL, 6 mol %) and UHP (0.28 g, 3.0mmol) were added and the mixture stirred at 10° C. After a further 72 hthe mixture was quenched with 5% aq. sodium bisulfite (20 mL). Themixture was diluted with EtOAc (80 mL), 5% aq. sodium bisulfite (50 mL)and 5% aq. sodium chloride (50 mL). The aqueous phase was extracted withEtOAc (80 mL), and the combined organics washed with 5% aq. sodiumchloride (50 mL), dried over sodium sulfate and concentrated in vacuo.The residue was purified by column chromatography on silica gel(heptane-EtOAc) to give the desired product (3.9 g, 73%) as an off-whitesolid. ¹H NMR (700 MHz, CDCl₃): δ=6.11 (1H, s, C4-CH), 3.46 (1H, d,J=3.9, C6-CH), 3.33 (1H, d, J=3.8, C7-CH), 2.55 (1H, ddd, J=5.6, 14.2,18.1, C2-CH_(a)H_(b)), 2.48-2.45 (1H, m, C2-CH_(a)H_(b)), 2.39 (1H, dd,J=3.8, 16.7, C22-CH_(a)H_(b)), 2.23 (1H, dd, J=7.6, 16.8,C22-CH_(a)H_(b)), 2.01-1.91 (4H, m, C1-CH_(a)H_(b), C12-CH_(a)H_(b),C15-CH_(a)H_(b), C16-CH_(a)H_(b)), 1.88 (1H, td, J=10.9, 1.3, C8-CH),1.84-1.80 (1H, m, C20-CH), 1.72 (1H, td, J=5.2, 13.9, C1-CH_(a)H_(b)),1.56-1.49 (2H, m, C11-CH_(a)H_(b), C14-CH), 1.38-1.21 (6H, m, C9-CH,C11-CH_(a)H_(b), C12-CH_(a)H_(b), C15-CH_(a)H_(b), C16-CH_(a)H_(b),C17-CH), 1.18 (3H, d, J=6.8, C21-CH₃), 1.10 (3H, s, C19-CH₃), 0.77 (3H,s, C18-CH₃); ¹³C NMR (176 MHz, CDCl₃): δ=198.3, 162.5, 131.2, 118.9,54.6, 54.5, 52.5, 51.2, 43.2, 40.5, 38.9, 35.5, 34.6, 34.1, 33.8, 33.7,28.2, 24.8, 23.6, 19.8, 19.3, 17.2, 11.9.

D. Synthesis of (6β, 7α,20R)-cyanomethyl-6-ethyl-7-hydroxy-4-pregnen-3-one

THF (17 mL) was charged to the reaction vessel, followed by 0.5 M zincchloride in THF (16.8 mL), and the mixture cooled to −15° C. 1MEthylmagnesium bromide in TBME (16.8 mL) was added dropwise over ca. 1h, maintaining the temperature <−7° C. Copper (I) chloride (92 mg, 0.93mmol) was charged to the reaction mixture.(20R)-Cyanomethyl-6,7-α-epoxy-4-pregnen-3-one (3.3 g, 9.3 mmol) wasdissolved in THF (19 mL) and charged dropwise to the reaction mixture,maintaining the temperature <−7° C. The mixture was stirred at −15° C.upon complete addition. After 1 h a second portion of 1M ethylmagnesiumbromide in TBME (17 mL) was added dropwise. The mixture was stirred at−15° C. After a further 30 min the mixture was quenched with sat. aq.ammonium chloride (3 mL) and allowed to warm to 15° C. The precipitatewas removed by filtration and rinsed with TBME (50 mL). The filtrate waswashed with sat. aq. ammonium chloride (3×50 mL) and 5% aq. sodiumchloride (25 mL), dried over Na₂SO₄, filtered and concentrated in vacuoto afford (6β, 7α, 20R)-cyanomethyl-6-ethyl-7-hydroxy-4-pregnen-3-one(3.2 g, 89%) as an off-white solid which was used without furtherpurification.

¹H NMR (700 MHz, CDCl₃): δ=5.78 (1H, s), 3.73 (1H, t, J=1.6), 2.48 (1H,ddd, J=17.5, 15.0, 4.9), 2.40-2.36 (2H, m), 2.31 (1H, ddd, J=8.7, 6.9,1.9), 2.23 (1H, dd, J=16.7, 7.4), 2.03 (1H, ddd, J=13.4, 5.1, 2.3), 1.99(1H, dt, J=12.7, 3.4), 1.95-1.90 (1H, m), 1.83-1.76 (3H, m), 1.70 (1H,td, J=5.7, 2.1), 1.63-1.44 (6H, m), 1.37-1.16 (5H, m), 1.22 (3H, s),1.18 (3H, d, J=6.7), 0.92 (3H, t, J=7.4), 0.76 (3H, s); ¹³C NMR (176MHz, CDCl₃): δ=199.1, 170.3, 128.7, 118.9, 72.1, 55.3, 54.8, 50.0, 44.2,42.6, 38.9, 38.3, 37.5, 35.6, 34.1, 33.6, 28.0, 26.3, 24.8, 23.6, 20.8,19.7, 19.3, 12.8, 11.9.

E. Synthesis of (5β, 6β, 7α,20R)-cyanomethyl-6-ethyl-7-hydroxy-pregna-3-one

(6β, 7α, 20R)-Cyanomethyl-6-ethyl-7-hydroxy-4-pregnen-3-one (3.1 g, 8.1mmol) was dissolved in DMF (54.5 mL) and 10% Pd/C charged (0.79 g of a45% dispersion in water). The mixture was degassed and filled withhydrogen. After 18 h 30 min the mixture was degassed and filled withargon, filtered and rinsed with TBME (3×60 mL). The filtrate wasre-filtered and rinsed with TBME (2×50 mL). The filtrate was washed with5% aq. sodium chloride (100 mL), and the aqueous phase re-extracted withTBME (100 mL). The combined organic phases were washed with 5% aq.sodium chloride (2×100 mL) and concentrated. The residue was purified byflash chromatography (heptane-EtOAc) to afford (5β, 6β, 7α,20R)-cyanomethyl-6-ethyl-7-hydroxy-pregna-3-one (2.5 g, 80%) as anoff-white solid.

¹H NMR (700 MHz, CDCl₃): δ=3.69 (1H, s), 3.38 (1H, dd, J 15.5, 13.4),2.39-2.34 (2H, m), 2.25 (1H, dd, J=16.7, 7.4), 2.14-2.08 (2H, m),2.04-1.98 (2H, m), 1.94-1.90 (2H, m), 1.83-1.80 (2H, m), 1.76-1.74 (1H,m), 1.64 (1H, td, J=11.2, 2.7), 1.60-1.54 (2H, m), 1.51-1.40 (4H, m),1.38-1.25 (4H, m), 1.21-1.15 (1H, m), 1.18 (3H, d, J=6.7), 1.05 (3H, s),0.94 (3H, t, J=7.1), 0.74 (3H, s); ¹³C NMR (176 MHz, CDCl₃): δ=213.7,118.9, 71.7, 54.9, 50.0, 49.9, 47.0, 46.7, 42.7, 39.1, 37.7, 36.3, 35.9,35.7, 34.0, 33.6, 28.1, 27.6, 24.8, 24.4, 23.7, 20.7, 19.3, 13.9, 11.9.

F. Synthesis of (5β, 6β, 20R)-cyanomethyl-6-ethyl-7-oxo-pregna-3-one

(5β, 6β, 7α, 20R)-Cyanomethyl-6-ethyl-7-hydroxy-pregna-3-one (2.4 g, 6.3mmol) was dissolved in DCM (60.5 mL) and cooled to 0° C. Dess-Martinperiodinane (DMP, 4.8 g, 11.3 mmol) was added over 1 minute. Thereaction mixture was stirred at 0° C. After 1 h a second portion of DMP(1.6 g, 3.8 mmol) was added. After 2 h a third portion of DMP (1.6 g,3.8 mmol) was added. After 3 h a fourth portion of DMP (0.5 g, 1.3 mmol)was added. After 3 h 45 min the mixture was diluted with 10% aq.Na₂S₂O₃/5% aq. NaHCO₃ (120 mL) and TBME (90 mL) and stirred vigorously.The phases were separated and the aqueous phase re-extracted with TBME(60 mL). The combined organic phases were concentrated and purified byflash chromatography (heptane-EtOAc) to afford (5β, 6β,20R)-cyanomethyl-6-ethyl-7-oxo-pregna-3-one (1.8 g, 75%) as an off-whitesolid.

¹H NMR (700 MHz, CDCl₃): δ=2.43 (1H, t, J=11.4), 2.38 (1H, dd, J=16.7,3.6), 2.30-2.20 (5H, m), 2.04 (1H, dt, J=12.7, 3.1), 2.01 (1H, dt,J=9.4, 4.7), 1.94-1.76 (7H, m), 1.66 (1H, ddd, J=14.3, 9.7, 4.3),1.58-1.52 (4H, m), 1.33-1.21 (4H, m), 1.18 (3H, d, J=6.7), 1.15 (3H, s),0.85 (3H, t, J=7.4), 0.75 (3H, s); ¹³C NMR (176 MHz, CDCl₃): δ=214.4,211.5, 118.8, 57.1, 54.0, 50.1, 48.4, 47.2, 44.7, 43.6, 43.2, 38.8,35.8, 35.2, 34.9, 33.5, 28.1, 24.8, 24.5, 23.7, 23.4, 21.6, 19.3, 12.6,12.3.

G. Synthesis of (3α, 5β, 6β, 20R)-cyanomethyl-6-ethyl-7-oxo-pregnane

Sodium borohydride (20 mg, 0.52 mmol) was suspended in isopropanol (0.8mL) and cooled to −20° C. (5β, 6β,20R)-cyanomethyl-6-ethyl-7-oxo-pregna-3-one (200 mg, 0.52 mmol) wasdissolved in ethyl acetate (1.7 mL) and TBME (1.2 mL) and added dropwiseto the cold borohydride suspension. The mixture was stirred at −20° C.for 45 min, then quenched by addition of 0.7 M sulfuric acid (1.4 mL)and allowed to warm to 18° C. The mixture was diluted with water (10 mL)and TBME (10 mL) and the phases separated. The aqueous phase wasre-extracted with TBME (10 mL) and the combined organic extracts washedwith 5% aq. sodium chloride (10 mL). The organic phase was concentratedand purified by flash chromatography to afford (3α, 5β,6β,20R)-cyanomethyl-6-ethyl-7-oxo-pregnane (113 mg, 56%, containing 10%3β-OH) as a pale yellow syrup.

¹H NMR (700 MHz, CDCl₃): δ=4.00-3.99 (0.1H, m, H-3_(3β-OH)), 3.68-3.53(0.9H, m, H-3_(3α-OH)), 2.57 (1H, dd, J=11.6, 11.1), 2.38 (1H, dd,J=16.7, 3.7), 2.23-2.20 (2H, m), 1.99-1.87 (5H, m), 1.83-1.64 (6H, m),1.55-1.45 (3H, m), 1.31-1.18 (7H, m), 1.22 (3H, s), 1.17 (3H, d, J=6.7),0.99-0.93 (1H, m), 0.84 (3H, t, J=7.3), 0.70 (3H. s); ¹³C NMR (176 MHz,CDCl₃): δ=215.3 (3α-C═O). 119.0 (CN), 70.5, 62.1, 54.0, 49.6, 48.7,45.5, 42.8, 42.6, 39.8, 38.5, 35.6, 35.4, 33.6, 29.5, 28.2, 26.6, 26.0,24.8, 24.8, 21.3, 19.4, 13.1, 12.2.

H. Synthesis of (3α, 5β, 6α)-6-ethyl-7-oxo-24-nor-lithocholic Acid

(3α, 5β, 6β, 20R)-cyanomethyl-6-ethyl-7-oxo-pregnane (65 mg, 0.17 mmol)was dissolved in methanol (3 mL) and 30% w/v potassium hydroxidesolution (3 mL) and heated to reflux for 4 days. The mixture was cooledin an ice bath and 6M hydrochloric acid added to pH 8 (2 mL). Ethylacetate (10 mL) was added, followed by 6M HCl to pH 1 (0.5 mL). Themixture was allowed to warm to 18° C. and the phases separated. Theorganic phase was washed with 5% aq. sodium chloride (20 mL) andconcentrated to afford (3α, 5β, 6α)-6-ethyl-7-oxo-24-nor-lithocholicacid (69 mg, quantitative) as a pale yellow syrup. ¹H NMR (700 MHz,CDCl₃): δ=3.56-3.52 (1H, m), 2.69 (1H, q, J=6.2), 2.48 (1H, dd, J=15.0,3.3), 2.36 (1H, t, J=11.3), 2.22-2.17 (1H, m), 2.05-2.02 (1H, m), 1.99(1H, dt, J=12.8, 3.3), 1.94-1.87 (2H, m), 1.84-1.69 (6H, m), 1.51-1.44(3H, m), 1.32-1.09 (6H, m), 1.22 (3H, s), 1.03 (3H, d, J=6.5), 0.98-0.92(1H, ddd, J=24.4, 12.3, 6.3), 0.86 (1H, q, J=12.6), 0.80 (3H, t, J=7.4),0.69 (3H, s); ¹³C NMR (176 MHz, CDCl₃): δ=212.9, 178.8, 71.2, 54.8,52.0, 50.7, 49.9, 49.0, 43.7, 72.7, 41.2, 38.9, 35.7, 34.2, 33.5, 31.7,29.8, 28.4, 24.6, 23.5, 21.8, 19.6, 18.8, 12.1, 12.0.

Alternatively, the product of step H can be converted to a compound ofgeneral formula (XXI) in which R^(4′) is C(O)OH by reduction, forexample with sodium borohydride.

As a person of skill in the art would appreciate, the synthetic routeshown in Scheme 5 could be adapted by conversion of the nitrile group toa carboxylic acid at an earlier stage followed, if necessary, byprotection of the carboxylic acid group, for example as an ester.

Example 6—Preparation of a Compound of General Formula (IF) with anAldehyde Side Chain

Scheme 7 illustrates a method for converting a compound of generalformula (II) with an aldehyde side chain to a compound of generalformula (IF) with an aldehyde side chain. The first step of the methodis to protect the aldehyde as a dioxolane group. The compound of generalformula (II) is then converted sequentially to compounds of generalformulae (IA), (IB), (IC), (ID), (IE) and (IF) using the reagents shownin Scheme 7, still with the aldehyde protected. The protection is thenremoved by treatment with acid.

A. Synthesis of (6β, 7α,20S)-20-(ethylenedioxymethyl)-6-ethyl-7-hydroxy-pregna-4-en-3-one

A solution of 0.5M ZnCl₂ in THF (3.1 mL) and THF (4 vol, 4 mL) wascooled to −15° C. and a solution of 1M EtMgBr in TBME (4.7 mL) was addeddropwise over 10 mins, maintaining the temperature below −12° C. CuCl(13 mg, 0.13 mmol) was then charged in one portion followed by thedropwise addition of a solution of (6α, 7α,20S)-6,7-epoxy-20-(ethylenedioxymethyl)-pregna-4-en-3-one from Example1F (1.0 g, 2.6 mmol) in THF (8 vol, 8 mL) over 16 mins, maintaining thetemperature below −12° C. The reaction was stirred at −15° C. for 40mins (TLC, eluant 1:1 EtOAc:Heptane; visualized with Cerium AmmoniumMolybdate stain), warmed to ambient temperature and quenched by thedropwise addition of sat. aq. NH₄Cl (2.5 vol, 2.5 mL). The reactionmixture was then diluted with EtOAc (50 mL) and washed with sat. aq.NH₄Cl (2×50 mL) and water (2×50 mL). The organic phase was dried overNa₂SO₄, filtered and concentrated in-vacuo at 40° C. Purification bycolumn chromatography gave (6β, 7α,20S)-20-(ethylenedioxymethyl)-6-ethyl-7-hydroxy-pregna-4-en-3-one as anoff white crystalline solid (1.06 g). ¹H NMR (700 MHz, CDCl₃): δ=5.78(1H, s), 4.85 (1H, d, J=2.0), 3.94 (2H, m), 3.89 (2H, m), 3.74 (1H, m),2.46 (1H, m), 2.37 (1H, m), 2.31 (1H, m), 2.06-1.93 (3H, m), 1.85-1.68(4H, m), 1.59 (3H, s), 1.58-1.25 (6H, m), 1.25 (1H, m), 1.22 (3H, s),1.18 (1H, m), 0.95 (3H, d, J=6.7), 0.91 (3H, t, J=7.4), 0.75 (3H, s).¹³C NMR (176 MHz, CDCl₃): δ=199.1, 170.4, 128.7, 106.0, 72.3, 65.2,65.1, 55.1, 52.3, 49.6, 44.4, 42.9, 39.3, 39.1, 38.3, 37.5, 35.7, 34.1,27.3, 26.4, 23.9, 20.9, 19.7, 12.8, 11.7, 11.6.

B. Synthesis of (5β, 6β, 7α,20S)-20-(ethylenedioxymethyl)-6-ethyl-7-hydroxy-pregna-3-one

5% Pd on CaCO₃ (90 mg, 0.2 mass eq) was charged to a flask under argon,followed by a solution of (6β, 7α,20S)-20-(ethylenedioxymethyl)-6-ethyl-7-hydroxy-pregna-4-en-3-one (450mg, 1.1 mmol) in DMF (3 vol, 2.25 mL) and MeCN (6 vol, 84.5 mL). Theflask was purged with argon, then and stirred at ambient temperature.After 24 h (TLC, eluant 1:1 EtOAc:Heptane; visualized with CeriumAmmonium Molybdate stain) the reaction mixture was purged with argon andthen filtered through a PTFE 0.45 μm filter. The filter was washed withEtOAc (2×25 mL). The organic phase was washed with water (3×25 mL),dried over Na₂SO₄, filtered and concentrated in-vacuo at 40° C.Purification by column chromatography gave (5β, 6β, 7α,20S)-20-(ethylenedioxymethyl)-6-ethyl-7-hydroxy-pregna-3-one as an offwhite crystalline solid (167 mg). ¹H NMR (700 MHz, CDCl₃): δ=4.85 (1H,d, J=2.0), 3.95 (2H, m), 3.85 (2H, m), 3.70 (1H, s), 3.37 (1H, dd,J=13.5, 15.5), 2.37 (1H, m), 2.11 (2H, m), 2.04-1.91 (4H, m), 1.81 (2H,m), 1.62-1.65 (3H, m), 1.55-1.40 (8H, m), 1.31-1.25 (2H, m), 1.18 (1H,m), 1.05 (3H, s), 0.95 (6H, m), 0.72 (3H, s). ¹³C NMR (176 MHz, CDCl₃):δ=213.8, 106.0, 72.0, 65.2, 65.0, 52.4, 49.7, 49.6, 47.0, 46.8, 43.0,39.3, 37.7, 36.3, 36.1, 35.8, 34.1, 31.9, 27.7, 27.4, 24.4, 24.0, 22.7,20.8, 13.9, 11.6.

C. Synthesis of (5β, 6β,20S)-20-(ethylenedioxymethyl)-6-ethyl-pregna-3,7-dione

To a solution of (5β, 6β, 7α,20S)-20-(ethylenedioxymethyl)-6-ethyl-7-hydroxy-pregna-3-one (110 mg,0.25 mmol) in CH₂Cl₂ (25 vol, 2.75 mL) under argon was added Dess-Martinperiodinane (127 mg, 0.3 mmol). After 30 minutes (TLC, eluant 1:1EtOAc:Heptane; visualized with Cerium Ammonium Molybdate stain) thereaction mixture was diluted with 10 EtOAc and 10% Na₂S₂O₃/2% NaHCO₃ andstirred for 1 h. The phases were separated and the aqueous extractedwith EtOAc (10 mL). The combined organic phases were washed with 1M aq.NaOH (10 mL), dried over Na₂SO₄, filtered and concentrated in-vacuo at40° C. to give crude (5β, 6β,20S)-20-(ethylenedioxymethyl)-6-ethyl-pregna-3,7-dione as a white solid(104 mg). ¹H NMR (700 MHz, CDCl₃): δ=4.85 (1H, d, J=2.0), 3.94 (2H, m),3.84 (2H, m), 2.42 (1H, t, J=11.4), 2.32-2.19 (4H, m), 2.06 (1H, m),2.02-1.75 (8H, m), 1.65 (1H, m), 1.59-1.39 (6H, m), 1.29-1.17 (2H, m),1.15 (3H, s), 0.94 (3H, d, J=6.7), 0.84 (3H, t, J=7.3), 0.73 (3H, s).¹³C NMR (176 MHz, CDCl₃): δ=214.6, 211.6, 105.9, 65.2, 65.0, 57.1, 51.5,49.8, 48.4, 47.4, 44.9, 43.6, 43.4, 39.2, 39.0, 35.8, 35.3, 34.9, 27.4,24.8, 23.8, 23.4, 21.7, 12.6, 12.0, 11.7.

D. Synthesis of (5β, 6α,20S)-20-(ethylenedioxymethyl)-6-ethyl-pregna-3,7-dione

A solution of (5β, 6β,20S)-20-(ethylenedioxymethyl)-6-ethyl-pregna-3,7-dione (100 mg, 0.3mmol) in MeOH (20 vol) was warmed to 50° C. and aq. 0.5M NaOH (0.65mmol) was added. After 16 h (TLC, eluant 1:1 EtOAc:Heptane; visualizedwith Cerium Ammonium Molybdate stain) the reaction was diluted withEtOAc (10 mL), washed with water (2×10 mL) and then 5% aq. NaCl (1×10mL). The combined organic phases were washed with 1M aq. NaOH (10 mL),dried over Na₂SO₄, filtered and concentrated in-vacuo at 40° C. to give(5β, 6α, 20S)-20-(ethylenedioxymethyl)-6-ethyl-pregna-3,7-dione as aclear oil (80 mg). ¹H NMR (700 MHz, CDCl₃): δ=4.85 (1H, d, J=2.0), 3.93(2H, m), 3.84 (2H, m), 2.74 (1H, q, J=4.6), 2.47 (1H, t, J=11.3),2.30-2.16 (4H, m), 2.10-2.02 (3H, m), 1.98 (1H, m), 1.91-1.79 (3H, m),1.72 (5H, m), 1.47-1.37 (2H, m), 1.33 (3H, s), 1.23 (1H, m), 1.07 (1H,m), 0.98 (1H, m), 0.94 (3H, d, J=6.7), 0.81 (3H, t, J=7.4), 0.71 (3H,s). ¹³C NMR (176 MHz, CDCl₃): δ=212.0, 210.6, 105.9, 65.2, 65.0, 52.3,52.2, 51.3, 50.0, 48.4, 43.7, 42.9, 39.1, 38.7, 38.3, 36.7, 35.9, 35.5,27.5, 24.7, 22.9, 22.2, 18.6, 11.9, 11.8, 11.7.

E. Synthesis of (3α, 5β, 6α,20S)-6-ethyl-3-hydroxy-20-(ethylenedioxymethyl)-pregna-7-one

NaBH₄ (80 mg, 0.2 mmol) in IPA (1.6 mL) was cooled to −15° C. (5β, 6α,20S)-20-(ethylenedioxymethyl)-6-ethyl-pregna-3,7-dione (80 mg, 0.2 mmol)in EtOAc (1.6 mL) was added dropwise over 10 mins. After 30 mins (TLC,eluant 1:1 EtOAc:Heptane; visualized with Cerium Ammonium Molybdatestain) the reaction was warmed to ambient temperature and quenched bythe addition of 0.7M aq. H₂SO₄ (7 vol) dropwise over 5 mins. Thereaction mixture was diluted with EtOAc (10 mL) and the organic phasewas washed with water (3×5 mL) and 5% aq. NaCl (1×5 mL). The organicphase was dried over Na₂SO₄, filtered and concentrated in-vacuo at 40°C. to give (3α, 5β, 6α,20S)-6-ethyl-3-hydroxy-20-(ethylenedioxymethyl)-pregna-7-one as a clearoil (60 mg). ¹H NMR (700 MHz, CDCl₃): δ=4.85 (1H, d, J=1.9), 3.93 (2H,m), 3.84 (2H, m), 3.52 (1H, m), 2.69 (1H, dd, J=5.7, 12.9), 2.21 (1H,m), 2.0-1.92 (2H, m), 1.86-1.67 (8H, m), 1.51-1.34 (6H, m), 1.25 (2H,m), 1.21 (3H, s), 1.20-1.10 (3H, m), 0.93 (3H, d, J=6.7), 0.88 (1H, m),0.80 (3H, t, J=7.4), 1.66 (3H, s). ¹³C NMR (176 MHz, CDCl₃): δ=212.7,106.0, 71.2, 65.3, 65.0, 52.0, 51.3, 50.7, 50.0, 48.5, 43.7, 43.0, 39.2,38.8, 35.7, 34.3, 31.8, 29.9, 27.6, 24.9, 23.5, 21.9, 18.8, 12.0, 11.9,11.7;

F. Synthesis of (3α, 5β, 6α, 7α,20S)-6-ethyl-3,7-dihydroxy-20-(ethylenedioxymethyl)-pregnane

To a solution (3α, 5β, 6α,20S)-6-ethyl-3-hydroxy-20-(ethylenedioxymethyl)-pregna-7-one (60 mg,0.14 mmol) in THF (5 mL) and water (1.25 mL) at 0° C., was added NaBH₄(53 mg, 1.4 mmol) in one portion. After 2 h (TLC, eluant 1:1EtOAc:Heptane; visualized with Cerium Ammonium Molybdate stain) thereaction was allowed to warm up to ambient temperature and was quenchedby the addition of 1:1 MeOH:H₂O (2 mL), followed by 2M aq. H₂SO₄ (1 mL)dropwise over 5 mins. The reaction mixture was diluted with EtOAc (20mL) and washed with water (3×20 mL). The aqueous phase was extractedwith EtOAc (20 mL) and the combined organic phases washed with 5% aq.NaCl (1×5 mL). The organic phase was dried over Na₂SO₄, filtered andconcentrated in-vacuo at 40° C. to give (3α, 5β, 6α, 7α,20S)-6-ethyl-3,7-dihydroxy-20-(ethylenedioxymethyl)-pregnan as a clearoil (58 mg). ¹H NMR (700 MHz, CDCl₃): δ=4.85 (1H, d, J=2.0), 3.94 (2H,m), 3.84 (2H, m), 3.40 (1H, m), 2.00-1.91 (2H, m), 1.80-1.75 (5H, m),1.70-1.63 (2H, m), 1.61-1.56 (1H, m), 1.53-1.12 (15H, m), 1.01 (1H, m),0.94 (3H, d, J=6.7), 0.90 (5H, m), 0.67 (3H, s). ¹³C NMR (176 MHz,CDCl₃): δ=104.9, 71.2, 69.7, 64.1, 63.9, 51.1, 48.8, 44.1, 41.9, 40.0,39.0, 38.3, 38.2, 34.4, 34.3, 32.8, 32.1, 29.5, 26.3, 22.8, 22.0, 21.1,19.6, 10.5, 10.4, 10.4.

G. Synthesis of (3α, 5β, 6α,7α)-6-ethyl-3,7-dihydroxy-23,24-dinor-cholane-22-al

(3α, 5β, 6α, 7α, 20S)-6-ethyl-3,7-dihydroxy-20-(ethylenedioxymethyl)-pregnan (58 mg, 0.14 mmol) in MeCN (1mL, 17 vol), H₂O (0.29 mL, 5 vol) and TFA (0.29 mL, 5 vol) was heated toreflux. After 2 h (TLC, eluant 1:1 EtOAc:Heptane; visualized withAnisaldehyde stain) the reaction mixture was poured onto 5% aq. NaHCO₃(30 mL) and diluted with CH₂Cl₂ (10 mL). After stirring for 15 minutesthe phases were separated and the aqueous phase extracted with CH₂Cl₂(2×100 mL). The combined organic phases were dried over Na₂SO₄, filteredand concentrated in-vacuo at 40° C. to give (3α, 5β, 6α,7α)-6-ethyl-3,7-dihydroxy-23,24-dinor-cholane-22-al as a mixture of C20epimers as a clear oil (51 mg). NMR data matches an authentic sample of(3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-23,24-dinor-cholane-22-al.

The compound of general formula (IF) with the aldehyde side chain canthen be converted to a compound of general formula (XXI) in which—YR^(4a) is C(O)OH by oxidation using any appropriate method. In onesuch method, the aldehyde could be directly oxidised to the acid using aJones reaction or KMnO₄. Alternatively, if chain extension is required,an olefination reaction followed by saponification will provide acompound in which R^(4a) is C(O)OH but in which Y has been extended asshown in Example 7.

Example 7—Preparation of a Compound of General Formula (XXI) ViaCompounds of General Formula (I) with OH and Aldehyde Side Chain

Scheme 8 below shows a method for the conversion of a compound ofgeneral formula (II) in which —YR⁴ is —CH₂OH to a compound of generalformula (XXI) in which —YR^(4a) is CH₂CH₂C(O)OH

A. Synthesis of (6β, 7α,20S)-20-acetoxymethyl-6-ethyl-7-hydroxy-pregna-4-en-3-one

A solution of 0.5 M ZnCl₂ in THF (20.2 mL) was charged to a reactionvessel under argon followed by THF (4 vol, 26 mL) and cooled to −15° C.A solution of 1 M EtMgBr in TBME (27 mL) was charged over 10 mins whilstmaintaining the temperature below −12° C. CuCl (84 mg, 0.84 mmol) wasthen charged in one portion. (6α, 7α,20S)-6,7-epoxy-20-acetoxymethyl-pregna-4-en-3-one (6.5 g, 16.8 mmol) inTHF (8 vol, 16 mL) was charged to the reaction vessel over 16 mins whilemaintaining the temperature below −12° C. and the reaction warmed toambient temperature and stirred for 90 mins. The reaction mixture wasquenched by the dropwise addition of sat. aq. NH₄Cl (2.5 vol, 17 mL).The reaction mixture was filtered and the filtrate washed with sat. aq.NH₄Cl (2×50 mL) and 5% aq. NaCl (2×50 mL). The organic phase was driedover Na₂SO₄, filtered and concentrated in-vacuo at 40° C. The crude (6β,7α, 20S)-20-acetoxymethyl-6-ethyl-7-hydroxy-pregna-4-en-3-one (6.7 g)was taken on to the next step with no further purification. ¹H NMR (700MHz, CDCl₃): δ=5.77 (1H, s), 4.07 (1H, dd, J=10.6, 3.1), 3.79 (1H, dd,J=10.6, 7.4), 3.74 (1H, s), 2.47 (1H, m), 2.37 (1H, m), 2.32 (1H, t,J=8.1), 2.05 (3H, s), 2.04-1.98 (3H, m), 1.90-1.65 (5H, m), 1.60-1.35(7H, m), 1.30-1.15 (6H, m), 1.02 (3H, d, J=6.6), 0.91 (3H, t, J=7.3),0.76 (3H, s); ¹³C NMR (176 MHz, CDCl₃): δ=199.2, 171.4, 170.9, 128.5,72.1, 69.4, 55.3, 52.6, 49.9, 44.2, 42.6, 39.0, 38.3, 37.4, 35.8, 35.6,34.1, 27.6, 26.3, 23.7, 21.0, 20.8, 19.7, 17.1, 12.8, 11.9.

B. Synthesis of (5β, 6β, 7α,20S)-20-acetoxymethyl-6-ethyl-7-hydroxy-pregna-3-one

5% Pd/CaCO₃ (274 mg, 0.2 mass eq) was charged to a flask under argon.(6β, 7α, 20S)-20-acetoxymethyl-6-ethyl-7-hydroxy-pregna-4-en-3-one (1.37g, 3.3 mmol) in DMF (3 vol, 4.1 mL) was charged followed by MeCN (6 vol,8.2 mL). The flask was purged with argon, purged with hydrogen andstirred at RT. After 24 h, the reaction mixture was purged with argonand filtered through Whatman® GF/B grade filter pad (glass fiber poresize 1 μm) filter pad. The solids were washed with EtOAc (2×25 mL). Thefiltrate was then washed with H₂O (3×30 mL), dried over Na₂SO₄ andconcentrated in-vacuo at 40° C. Purification by column chromatographygave (5β, 6β, 7α, 20S)-20-acetoxymethyl-6-ethyl-7-hydroxy-pregna-3-oneas an off white crystalline solid (0.96 g, 69%). ¹H NMR (700 MHz,CDCl₃): δ=4.08 (1H, dd, J=10.7, 3.4), 3.79 (1H, dd, J=10.7, 7.3), 3.71(1H, s), 3.36 (1H, dd, J=15.5, 13.5), 2.36 (1H, td, J=14.1, 4.6), 2.11(1H, m), 2.06 (3H, s), 2.03-1.10 (21H, m), 1.05 (3H, s), 1.03 (3H, d,J=6.6), 0.94 (3H, t, J=7.1), 0.73 (3H, s); ¹³C NMR (176 MHz, CDCl₃):δ=213.7, 171.4, 72.0, 69.5, 52.7, 49.9, 49.8, 47.0, 46.7, 42.8, 39.3,37.7, 36.3, 36.0, 35.8, 35.7, 34.2, 27.7, 27.6, 24.4, 23.9, 21.0, 20.8,17.2, 13.9, 11.8.

C. Synthesis of (5β, 6β,20S)-6-ethyl-3,7-dioxo-23,24-dinor-cholane-22-ol acetate [or (5β, 6β,20S)-20-acetoxymethyl-6-ethyl-pregna-3,7-dione]

(5β, 6β, 7α, 20S)-20-acetoxymethyl-6-ethyl-7-hydroxy-pregna-3-one (3.31g, 7.9 mmol) was dissolved in CH₂Cl₂ (25 vol, 83 mL) under argon andcooled to 0° C. Dess Martin periodane (4.0 g, 9.5 mmol) was charged inportions over 5 mins. After 20 mins the reaction was quenched by theaddition of 10% aq. Na₂SO₄/2% aq. NaHCO₃ (20 mL) and the mixture stirredfor 20 mins. The solution was diluted with EtOAc (100 mL) and H₂O (100mL). The aqueous layer was separated and extracted with EtOAc (100 mL).The combined organic layers were washed with 1M aq. NaOH (50 mL), then5% aq. NaCl (50 mL) and the resulting cloudy solution passed through asilica plug and washed with EtOAc (2×100 mL). Concentration in-vacuo at40° C. followed by purification by column chromatography gave (5β, 6β,20S)-6-ethyl-3,7-dioxo-23,24-dinor-cholane-22-ol acetate as a whitecrystalline solid (2.39 g, 73%). ¹H NMR (700 MHz, CDCl₃): δ=4.08 (1H,dd, J=10.7, 3.4), 3.79 (1H, dd, J=10.7, 7.4), 2.44 (1H, t, J=11.4),2.31-2.19 (4H, m), 2.05 (3H, s), 2.00 (1H, m), 1.92-1.71 (6H, m), 1.65(1H, m), 1.59-1.47 (3H, m), 1.39-1.17 (7H, m), 1.16 (3H, s), 1.03 (3H,d, J=6.7), 0.85 (3H, t, J=7.4), 0.75 (3H, s); ¹³C NMR (176 MHz, CDCl₃):δ=214.6, 211.6, 171.3, 69.4, 57.3, 52.0, 50.0, 48.5, 47.3, 44.9, 43.6,43.2, 39.0, 35.8, 35.7, 35.3, 35.0, 27.7, 24.7, 23.8, 23.5, 21.7, 21.0,17.2, 12.6, 12.2.

D. Synthesis of (5β, 6α,20S)-6-ethyl-3,7-dioxo-23,24-dinor-cholane-22-ol [or (5β, 6α,20S)-6-ethyl-20-hydroxymethyl-pregna-3,7-dione]

To a suspension of (5β, 6β,20S)-6-ethyl-3,7-dioxo-23,24-dinor-cholane-22-ol acetate (1.77 g, 4.2mmol) in EtOH (12 vol, 21.5 mL) at 50° C. was added dropwise 0.5M aq.NaOH (18.9 mL, 9.45 mmol). The reaction was heated at 50° C. for 16 h,then cooled to ambient temperature, diluted with H₂O (50 mL) andextracted with EtOAc (50 mL). The phases were separated and the aqueousphase extracted with EtOAc (2×50 mL). The combined organic phases werewashed with 5% aq. NaCl (2×50 mL), dried over Na₂SO₄, filtered andconcentrated in-vacuo at 40° C. Purification by column chromatographygave (5β, 6α, 20S)-6-ethyl-3,7-dioxo-23,24-dinor-cholane-22-ol as awhite crystalline solid (1.35 g, 86%). ¹H NMR (700 MHz, CDCl₃): δ=3.64(1H, dd, J=10.4, 2.9), 3.37 (1H, dd, J=10.3, 7.1), 2.69 (1H, m), 2.47(1H, t, J=11.3), 2.30-2.16 (5H, m), 2.10-2.03 (2H, m), 1.94-1.80 (3H,m), 1.72-1.49 (6H, m), 1.43 (1H, br.s), 1.33 (3H, s), 1.32-1.17 (3H, m),1.06 (3H, d, J=6.7), 0.98 (1H, m), 0.81 (3H, t, J=7.4), 0.71 (3H, s);¹³C NMR (176 MHz, CDCl₃): δ=212.1, 210.6, 67.8, 52.4, 52.2, 51.5, 50.0,48.7, 43.7, 42.7, 38.8, 38.6, 38.3, 36.7, 35.9, 35.5, 27.9, 24.7, 22.9,22.3, 18.6, 16.8, 12.2, 11.8.

E. Synthesis of (3α, 5β, 6α, 20S)-6-ethyl-3-hydroxy-7-oxo-23,24-dinor-cholane-22-ol [or (3α, 5β, 6α,20S)-6-ethyl-3-hydroxy-20-hydroxymethyl-pregna-7-one]

NaBH₄ (136 mg, 3.6 mmol) in IPA (6.5 vol, 9 mL) was cooled to −15° C.,then a solution of (5β, 6α,20S)-6-ethyl-3,7-dioxo-23,24-dinor-cholane-22-ol (1.35 g, 0.3.6 mmol) inEtOAc (6.5 vol, 9 mL) was added dropwise over 10 mins. After 20 mins thereaction was warmed to ambient temperature and quenched by the dropwiseaddition of 0.7M aq. H₂SO₄ (7 vol, 9.45 mL) over 10 mins. The reactionmixture was diluted with EtOAc (50 mL) and the organic phase washed withH₂O (3×50 mL) and 5% aq. NaCl (50 mL). The organic phase was dried overNa₂SO₄, filtered and concentrated in-vacuo at 40° C. Purification bycolumn chromatography and concentration in-vacuo at 40° C. gave (3α, 5β,6α, 20S)-6-ethyl-3-hydroxy-7-oxo-23,24-dinor-cholane-22-ol as a whitecrystalline solid (0.83 g, 61%). ¹H NMR (700 MHz, CDCl₃): δ=3.64 (1H,dd, J=10.5, 3.2), 3.53 (1H, m), 3.35 (1H, dd, J=10.4, 7.1), 2.69 (1H,m), 2.35 (1H, t, J=11.2), 2.20 (1H, m), 2.00 (1H, m), 1.92-1.67 (8H, m),1.57-1.43 (3H, m), 1.34-1.23 (2H, m), 1.23 (3H, s), 1.21-1.10 (4H, m),1.04 (3H, d, J=6.6), 0.98-0.83 (2H, m), 0.80 (3H, t, J=7.4), 0.67 (3H,s); ¹³C NMR (176 MHz, CDCl₃): δ=212.9, 71.2, 67.9, 52.0, 51.6, 50.7,50.0, 48.8, 43.7, 42.8, 38.9, 38.7, 35.7, 34.3, 31.8, 29.6, 27.9, 24.8,23.5, 21.9, 18.8, 16.8, 12.1, 12.0.

F. Synthesis of (3α, 5β, 6α, 7α,20S)-6-ethyl-3,7-dihydroxy-23,24-dinor-cholane-22-ol (or (3α, 5β, 6α,7α, 20S)-6-ethyl-3,7-dihydroxy-20-hydroxymethyl-pregnan)

(3α, 5β, 6α, 20S)-6-ethyl-3-hydroxy-7-oxo-23,24-dinor-cholane-22-ol(0.83 g, 2.2 mmol) in THF (30 mL) and water (7.5 mL) was cooled to 0° C.and NaBH₄ (830 mg, 22 mmol) added in 4 portions over 15 mins. After 2 hthe reaction was warmed to room temperature and quenched by the additionof 1:1 MeOH:H₂O (15 mL) followed by the dropwise addition of 2M aq.H₂SO₄ (11 mL) over 10 mins. The reaction mixture was diluted with EtOAc(100 mL) and washed with H₂O (100 mL). The aqueous phase was extractedwith EtOAc (3×100 mL) and the combined organic phases were washed with5% aq. NaCl (3×100 mL). The organic phase was dried over Na₂SO₄,filtered and concentrated in-vacuo at 40° C. to give (3α, 5β, 6α, 7α,20S)-6-ethyl-3,7-dihydroxy-23,24-dinor-cholane-22-ol as a white solid(0.53 g, 64%). ¹H NMR (700 MHz, MeOD): δ=3.64 (1H, s), 3.57 (1H, dd,J=10.6, 3.1), 3.30 (1H, m), 3.23 (1H, dd, J=10.5, 7.4), 2.00 (1H, m),1.90-1.70 (6H, m), 1.59 (1H, m), 1.57-1.44 (6H, m), 1.42-1.27 (5H, m),1.21 (2H, m), 1.13 (1H, m), 1.04 (3H, d, J=6.6), 1.00 (1H, m), 0.91 (3H,s), 0.90 (3H, t, J=7.7), 0.71 (3H, s); ¹³C NMR (176 MHz, MeOD): δ=71.7,69.7, 66.5, 52.5, 50.0, 45.5, 42.3, 41.7, 40.1, 39.5, 38.8, 35.3, 35.1,33.1, 32.9, 29.8, 27.5, 23.2, 22.3, 22.0, 20.5, 15.9, 10.9, 10.6.

G. Synthesis of (3α, 5β, 6α,7α)-6-ethyl-3,7-dihydroxy-23,24-dinor-cholane-22-al

(3α, 5β, 6α, 7α, 20S)-6-ethyl-3,7-dihydroxy-23,24-dinor-cholane-22-ol(421 mg, 1.11 mmol) in DMF (50 vol, 20 mL) was cooled to 0° C. DessMartin periodinane (473 mg, 1.12 mmol) was charged in portions. After2.5 h (TLC, eluant 7:3 EtOAc:Heptane; visualized with Cerium AmmoniumMolybdate stain), the reaction was quenched by the addition of 10% aq.NaHSO₃/2% aq. NaHCO₃ (5 mL) and the mixture stirred for 10 mins. Themixture was diluted with EtOAc (100 mL) and 5% NaCl (5 mL). The aqueouslayer was extracted with EtOAc (50 mL). The combined organic phases werewashed with 2M aq. NaOH (50 mL) and 5% aq. NaCl (4×50 mL), dried overNa₂SO₄, filtered and concentrated in-vacuo at 40° C. Purification bycolumn chromatograph gave (3α, 5β, 6α,7α)-6-ethyl-3,7-dihydroxy-23,24-dinor-cholane-22-al as a 3:1 mixturewith (5β, 6α, 7α)-6-ethyl-7-hydroxy-7-oxo-23,24-dinor-cholane-22-al(white foam, 230 mg). ¹H NMR (700 MHz, CDCl₃): δ=9.56 (1H, d, J=3.4),3.71 (1H, br. s), 3.44-3.36 (1H, m), 2.38-2.33 (1H, m), 1.94-1.86 (2H,m), 1.83-1.81 (2H, m), 1.80-1.78 (2H, m), 1.74-1.36 (10H, m), 1.34-1.18(8H, m), 1.14 (3H, d, J=6.8), 0.91 (3H, s), 0.88 (3H, t, J=7.07), 0.71(3H, s). ¹³C NMR (176 MHz, CDCl₃): δ=205.1, 72.3, 70.9, 51.0, 49.9,49.5, 45.1, 43.3, 41.2, 40.0, 39.3, 35.6, 35.5, 34.0, 33.4, 30.6, 27.1,24.1, 23.1, 22.2, 20.7, 13.5, 12.2, 11.6.

H. Synthesis of (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-22-cholen-24-oicAcid Ethyl Ester

The HWE reagent was prepared by dropwise addition of TEPA (262 μL, 1.32mmol) to NaOEt (91 mg, 1.3 mmol) in CH₂Cl₂ (2 mL) at 0° C. The reactionmixture was added dropwise over 10 minutes to a solution of (3α, 5β, 6α,7α)-6-ethyl-3,7-dihydroxy-23,24-dinor-cholane-22-al (199 mg, 0.528 mmol)in CH₂Cl₂ (4 mL) at 0° C. The reaction was warmed to ambient temperatureand stirred for 1 hour (TLC, eluant 1:1 EtOAc:Heptane; visualized withCerium Ammonium Molybdate stain). The mixture was diluted with H₂O (20mL) and CH₂Cl₂ (15 mL). The aqueous layer was separated and extractedwith CH₂Cl₂ (3×20 mL). The combined organic phases were dried overNa₂SO₄, filtered and concentrated. Purification by column chromatographygave (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-22-cholen-24-oic acid ethylester as a white foam (158 mg). The isolated product is a 4:1 mixture ofthe desired (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-22-cholen-24-oic acidethyl ester and (5β, 6α, 7α)-6-ethyl-7-dihydroxy-3-oxo-22-cholen-24-oicacid ethyl ester. ¹H NMR (700 MHz, CDCl₃): δ=6.83 (1H, dd, J=9.0, 15.6),5.73 (1H, d, J=15.3), 4.17 (2H, q, J=7.1), 3.69 (1H, m), 3.40 (1H, m),2.30-2.25 (1H, m), 1.92 (1H, m), 1.85-1.76 (2H, m), 1.76-1.62 (5H, m),1.59 (1H, m), 1.54-1.34 (7H, m), 1.29 (3H, t, J=7.1), 1.33-1.23 (6H, m),1.09 (3H, d, J=6.6), 0.90 (3H, s), 0.90 (3H, t, J=7.4), 0.68 (3H, s).¹³C NMR (176 MHz, CDCl₃): δ=167.1, 154.7, 119.0, 72.3, 70.8, 60.1, 54.9,50.4, 45.2, 43.0, 41.0, 40.1, 39.8, 39.5, 35.6, 35.5, 34.0, 33.3, 30.6,28.2, 23.7, 23.1, 22.2, 20.7, 19.3, 14.3, 12.1, 11.7.

I. (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-24-oic Acid Ethyl Ester

10% Palladium on Carbon (79 mg) was charged to a flask under argon. Asolution of (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-22-cholen-24-oic acidethyl ester (135 mg, 0.312 mmol) in EtOAc (51 vol, 7.0 mL) was chargedand purged with H₂. After 70 h (TLC, eluant 1:1 EtOAc:Heptane;visualized with Anisaldehyde stain) the reaction mixture was filteredthrough a 0.45 μm PTFE filter and the filter washed with EtOAc (10 mL).Concentration in-vacuo at 40° C. gave (3α, 5β, 6α,7α)-6-ethyl-3,7-dihydroxy-cholan-24-oic acid ethyl ester (134 mg) as a4:1 mixture with (5β, 6α, 7α)-6-ethyl-7-hydroxy-3-oxo-cholan-24-oic acidethyl ester. ¹H NMR (500 MHz, CDCl₃): δ=4.13 (2H, q, J=7.2), 3.46-3.37(1H, m), 2.41-2.32 (1H, m), 2.28-2.19 (1H, m), 1.89-1.76 (6H, m),1.76-1.57 (5H, m), 1.54-1.34 (12H, m), 1.27 (3H, t, J=7.1), 1.25-1.12(4H, m), 0.98-0.88 (9H, m), 0.68 (3H, s). ¹³C NMR (126 MHz, CDCl₃):δ=167.1, 154.7, 119.0, 72.3, 70.8, 60.1, 54.9, 50.4, 45.2, 43.0, 41.0,40.1, 39.8, 39.5, 35.6, 35.5, 34.0, 33.3, 30.6, 28.2, 23.7, 23.1, 22.2,20.7, 19.3, 14.3, 12.1, 11.7.

J. Synthesis of (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-24-oicAcid (Obeticholic Acid)

To (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-24-oic acid ethyl ester(118 mg, 0.272 mmol) in EtOH (34 vol, 4 mL) at 50° C., was added 0.5Maq. NaOH (1.2 mL, 0.61 mmol) dropwise. The reaction mixture was stirredat 50° C. for 2.5 h (TLC, eluent 1:1 EtOAc:Heptane; visualized withCerium Ammonium Molybdate stain) and then 0.5M aq. NaOH (1 mL, 0.5 mmol)was added. After 1 h, the reaction was quenched with 3M aq. HCl (2 mL).The aqueous phase was separated and extracted with EtOAc (3×15 mL). Thecombined organic phases were dried over Na₂SO₄, filtered andconcentrated in-vacuo at 40° C. Purification by column chromatographygave (3α, 5β, 6α, 7α)-6-ethyl-3,7-dihydroxy-cholan-24-oic acid (108 mg,white foam) as a 4:1 mixture with (5β, 6α,7α)-6-ethyl-7-hydroxy-3-oxo-cholan-24-oic acid. NMR data was consistentwith an authentic sample of OCA.

Alternatively, the product of step E can be converted to (3α, 5β,6α)-6-ethyl-3-hydrox-7-oxo-cholan-24-oic acid via a 7-oxo intermediatewith an aldehyde substituent on the side chain.

K. Synthesis of (3α, 5β,6α)-6-ethyl-3-hydroxy-7-oxo-23,24-dinor-cholane-22-al

To a solution of (3α, 5β, 6α,20S)-6-ethyl-3-hydroxy-7-oxo-23,24-dinor-cholane-22-ol (0.5 g, 1.33mmol) in CH₂Cl₂ (100 vol, 50 mL) at 0° C. under argon was charged inportions Dess Martin periodane (564 mg, 1.33 mmol) over 20 mins. After2.5 h (TLC, eluant 1:1 EtOAc:Heptane; visualized with Cerium AmmoniumMolybdate stain) the reaction was quenched by the addition of 10% aq.Na₂SO₄/2% NaHCO₃ (10 mL) and the mixture stirred for 10 mins. Thesolution was diluted with EtOAc (100 mL) and H₂O (100 mL). The aqueouslayer was separated and extracted with EtOAc (100 mL). The combinedorganic layers were washed with 1M aq. NaOH (50 mL), 5% aq. NaCl (100mL), dried over Na₂SO₄, filtered and concentration in-vacuo at 40° C.Purification by column chromatography gave (3α, 5β,6α)-6-ethyl-3-hydroxy-7-oxo-23,24-dinor-cholane-22-al as an opaque oil(229 mg) along with recovered starting material (144 mg). ¹H NMR (700MHz, CDCl₃): δ=9.57 (1H, d, J=3.4), 3.54 (1H, m), 2.69 (1H, dd, J=5.7,13.0), 2.35 (2H, m), 2.26 (2H, m), 1.97-1.90 (2H, m), 1.85-1.68 (7H, m),1.55-1.46 (4H, m), 1.41-1.34 (1H, m), 1.23 (3H, s), 1.22-1.15 (3H, m),1.12 (3H, d, J=6.9), 1.01 (1H, m), 0.87 (1H, m), 0.81 (3H, t, J=7.4),0.70 (3H, s). ¹³C NMR (176 MHz, CDCl₃): δ=212.4, 205.0, 71.1, 52.0,50.6, 50.3, 49.8, 49.2, 48.4, 43.7, 43.2, 38.7, 35.7, 34.3, 31.8, 29.8,27.1, 25.0, 23.5, 21.8, 18.8, 13.6, 12.5, 12.0

L. Synthesis of (3α, 5β, 6α)-6-ethyl-3-hydroxy-7-oxo-22-cholen-24-oicAcid Ethyl Ester

To a suspension of NaOEt (43 mg, 0.63 mmol) in CH₂Cl₂ (0.8 mL) at 0° C.was added dropwise TEPA and the solution warmed to ambient temperature.The TEPA/NaOEt mixture was then added dropwise to a solution of (3α, 5β,6α)-6-ethyl-3-hydroxy-7-oxo-23, 24-dinor-cholane-22-al (195 mg, 0.52mmol) in CH₂Cl₂ (4 mL) at 0° C. over 10 mins. The reaction was stirredat 0° C. for 1 h and then ambient temperature for 1 h. The reactionmixture was then re-cooled to 0° C. and a further aliquot of theTEPA/NaOEt mixture added. The reaction was stirred at 0° C. and after0.5 h (TLC, eluant 1:1 EtOAc:Heptane; visualized with Cerium AmmoniumMolybdate stain) H₂O (3 vol, 0.6 mL) was added and the reaction mixturewarmed to ambient temperature. The solution diluted with CH₂Cl₂ (10 mL)and washed with H₂O (10 mL). The aqueous layer was separated andextracted with CH₂Cl₂ (10 mL). The combined organic layers were washedwith 5% aq. NaCl (10 mL), dried over Na₂SO₄, filtered and concentrated.Purification by column chromatography gave (3α, 5β,6α)-6-ethyl-3-hydroxy-7-oxo-22-cholen-24-oic acid ethyl ester as anopaque oil (130 mg). ¹H NMR (700 MHz, CDCl₃): δ=6.82 (1H, dd, J=9.0,15.5), 5.72 (1H, d, J=15.5), 4.17 (2H, q, J=7.1), 3.53 (1H, m), 2.69(1H, dd, J=5.8, 13.0), 2.36 (1H, t, J=11.3), 2.26 (1H, m), 2.17 (1H, m),1.85-1.68 (9H, m), 1.47 (3H, m), 1.28 (3H, t, J=7.3), 1.27-1.23 (3H, m),1.22 (3H, s), 1.20-1.10 (3H, m), 1.08 (3H, d, J=6.7), 0.97-0.83 (2H, m),0.80 (3H, t, J=7.4), 0.68 (3H, s). ¹³C NMR (176 MHz, CDCl₃): δ=212.6,167.1, 154.5, 119.1, 71.1, 60.2, 54.0, 52.0, 50.6, 49.9, 48.9, 43.7,42.9, 39.5, 38.8, 35.7, 34.3, 31.8, 29.9, 28.2, 24.7, 23.5, 21.8, 19.4,18.8, 14.3, 12.4, 12.0.

M. Synthesis of (3α, 5β, 6α)-6-ethyl-3-hydroxy-7-oxo-cholan-24-oic AcidEthyl Ester

10% Palladium on Carbon (53 mg) was charged to a flask under argon. (3α,5β, 6α)-6-ethyl-3-hydroxy-7-oxo-22-cholen-24-oic acid ethyl ester (107mg, 0.24 mmol) dissolved in EtOAc (5.4 mL) was charged and the reactionpurged with argon and then H₂. After 16 h at ambient temperature (TLC,eluant 1:1 EtOAc:Heptane; visualized with Anisaldehyde stain) thereaction mixture was filtered through a 0.45 μm PTFE filter and thefilter washed with EtOAc (10 mL). Concentration in-vacuo at 40° C. gave(3α, 5β, 6α)-6-ethyl-3-hydrox-7-oxo-cholan-24-oic acid ethyl ester as aclear oil (86 mg). ¹H NMR (700 MHz, CDCl₃): δ=4.12 (2H, ddd, J=1.5, 7.1,14.2), 3.53 (1H, m), 2.69 (1H, dd, J=5.7, 13.0), 2.35 (2H, m), 2.20 (2H,m), 2.00-1.90 (2H, m), 1.85-1.66 (9H, m), 1.50-1.39 (4H, m), 1.36-1.29(2H, m), 1.25 (3H, t, J=7.1), 1.21 (3H, s), 1.20-1.08 (4H, m), 0.92 (3H,d, J=6.6), 0.90-0.82 (2H, m), 0.80 (3H, t, J=7.4), 0.65 (3H, s). ¹³C NMR(176 MHz, CDCl₃): δ=212.8, 174.3, 71.2, 60.2, 54.8, 52.0, 50.7, 49.9,49.0, 43.7, 42.7, 39.0, 35.7, 35.2, 34.3, 31.8, 31.3, 31.0, 29.9, 28.3,24.6, 23.5, 21.9, 18.8, 18.4, 14.3, 12.1, 12.0.

N. Synthesis of (3α, 5β, 6α)-6-ethyl-3-hydrox-7-oxo-cholan-24-oic Acid

To a solution of (3α, 5β, 6α)-6-ethyl-3-hydrox-7-oxoy-cholan-24-oic acidethyl ester (73 mg, 0.16 mmol) in EtOH (1 mL) at 50° C. was addeddropwise 0.5M aq. NaOH (0.72 mL, 0.36 mmol). The reaction was heated at50° C. for 1 h (TLC, eluent 1:1 EtOAc:Heptane; visualized with CeriumAmmonium Molybdate stain), quenched by the addition of 2M aq. HCl (1 mL)and then diluted with H₂O (10 mL) and EtOAc (10 mL). The phases wereseparated and the aqueous phase extracted with EtOAc (2×10 mL). Thecombined organic phases were washed with 5% aq. NaCl (2×10 mL), driedover Na₂SO₄, filtered and concentrated in-vacuo at 40° C. Purificationby column chromatography gave (3α, 5β,6α)-6-ethyl-3-hydrox-7-oxo-cholan-24-oic acid as an oil (54 mg). ¹H NMR(700 MHz, CDCl₃): δ=3.53 (1H, m), 2.69 (1H, dd, J=6.1, 12.9), 2.37 (2H,m), 2.25 (1H, m), 2.18 (1H, m), 2.0-1.89 (2H, m), 1.85-1.68 (7H, m),1.50-1.40 (4H, m), 1.38-1.23 (5H, m), 1.22 (3H, s), 1.20-1.09 (4H, m),0.93 (3H, d, J=6.6), 0.91-0.83 (2H, m), 0.80 (3H, t, J=7.4), 0.65 (3H,s) ¹³C NMR (176 MHz, CDCl₃): δ=213.0, 179.6, 71.2, 54.8, 52.0, 50.7,49.9, 49.0, 43.7, 42.7, 39.0, 35.7, 35.2, 34.3, 31.7, 31.0, 30.8, 29.8,28.3, 24.6, 23.5, 21.9, 18.8, 18.4, 12.1, 12.0.

Examples 1 to 7 illustrate how the various side chains YR⁴ of thecompounds of general formula (I) can be interconverted and how thecompounds of general formula (IA), (IB), (IC), (ID), (IE) and (IF) canbe converted to the required products of general formula (XXI).

The invention claimed is:
 1. A compound of general formula (I):

wherein:

is a carbon-carbon single or double bond; R¹ is C₁₋₄ alkyl optionallysubstituted with one or more substituents selected from halo, OR^(7a)and NR^(7a)R^(7b); where each of R^(7a) and R^(7b) is independentlyselected from H and C₁₋₄ alkyl; or R¹ together with R² forms an epoxidegroup; R² is selected from the group consisting of ═O, OH and aprotected OH or R² together with R¹ forms an epoxide group; R³ isselected from the group consisting of H, halo, OH and a protected OH;when

is a carbon-carbon double bond, Y is selected from the group consistingof a bond and an alkylene, alkenylene or alkynylene linker group havingfrom 1 to 20 carbon atoms and optionally substituted with one or moregroups R¹³; when

is a carbon-carbon single bond, Y is selected from the group consistingof a bond and an alkylene linker group having from 1 to 20 carbon atomsand optionally substituted with one or more groups R¹³; or Y, togetherwith R⁴ forms a group ═CH₂; each R¹³ is independently selected fromhalo, OR⁸ and NR⁸R⁹; where each of R⁸ and R⁹ is independently selectedfrom H and C₁₋₄ alkyl; R⁴ is selected from the group consisting of halo,CN, CH(OR¹⁰)(OR¹¹), CH(SR¹⁰)SR¹¹), NR¹⁰R¹¹, BR¹⁰R¹¹, C(O)CH₂N₂, —CH═CH₂,—C≡CH, CH[C(O)OR¹⁰]₂, CH(BR¹⁰R¹¹)², azide and a carboxylic acid mimeticgroup selected from —SO₂—NHR³⁰, C(O)NH—SO₂R³⁰, NHC(O)NH—SO₂R³⁰ andtetrazole optionally substituted with one or more substituents selectedfrom C₁₋₄ alkyl, halo, OH, O(C₁₋₄ alkyl), SO₂(C₁₋₄ alkyl), SO₂-phenyland SO₂-tolyl; where R³⁰ is selected from H, C₁₋₆ alkyl C₃₋₇ cycloalkylor aryl optionally substituted with C₁₋₄ alkyl, halo, OH, O(C₁₋₄ alkyl),SO₂(C₁₋₄ alkyl), SO₂-phenyl or SO₂-tolyl; where each R¹⁰ and R¹¹ isindependently selected from: a. hydrogen and b. C₁₋₂₀ alkyl, C₂₋₂₀alkenyl or C₂₋₂₀ alkynyl, any of which is optionally substituted withone or more substituents selected from halo, NO₂, CN, OR¹⁹, SR¹⁹,C(O)OR¹⁹, C(O)N(R¹⁹)₂, SO₂R¹⁹, SO₃R¹⁹, OSO₃R¹⁹, N(R¹⁹)₂ and a 6- to14-membered aryl or 5 to 14-membered heteroaryl group, either of whichis optionally substituted with one or more substituents selected fromC₁₋₆ alkyl, C₁₋₆ haloalkyl, halo, NO₂, CN, OR¹⁹, SR¹⁹, C(O)OR¹⁹,C(O)N(R¹⁹)₂, SO₂R¹⁹, SO₃R¹⁹, OSO₃R¹⁹ and N(R¹⁹)₂; and c. a 6- to14-membered aryl or 5 to 14-membered heteroaryl group either of which isoptionally substituted with one or more substituents selected from C₁₋₆alkyl, C₁₋₆ haloalkyl, halo, NO₂, CN, OR¹⁹, SR¹⁹, C(O)OR¹⁹, C(O)N(R¹⁹)₂,SO₂R¹⁹, SO₃R¹⁹, OSO₃R¹⁹ and N(R¹⁹)₂; and d. a polyethylene glycolresidue; or e. when R⁴ is CH(OR¹⁰)(OR¹¹), CH(SR¹⁰)(SR¹¹), NR¹⁰R¹¹,BR¹⁰R¹¹, CH[C(O)OR¹⁰]₂ or CH(BR¹⁰R¹¹)₂ an R¹⁰ and an R¹¹ group, togetherwith the atom or atoms to which they are attached, may combine to form a3 to 10-membered heterocyclic ring; each R¹⁹ is independently selectedfrom H, C₁₋₆ alkyl, C₁₋₆ haloalkyl and a 6- to 14-membered aryl or 5 to14-membered heteroaryl group either of which is optionally substitutedwith one or more substituents selected from halo, C₁₋₆ alkyl and C₁₋₆haloalkyl; or R⁵ is selected from the group consisting of H, OH and aprotected OH group; R⁶ is ═O; and salts thereof.
 2. The compoundaccording to claim 1 selected from the group consisting of: a compoundof general formula (IA):

wherein R³, Y, R⁴ and R⁵ are as defined for general formula (I); acompound of general formula (IB):

wherein R¹, R³, Y, R⁴ and R⁵ are as defined for general formula (I); acompound of general formula (IC):

wherein R¹, R³, Y, R⁴ and R⁵ are as defined for general formula (I); acompound of general formula (ID):

wherein R¹, R³, Y, R⁴ and R⁵ are as defined for general formula (I); anda compound of general formula (IE):

wherein R¹, R³, Y, R⁴ and R⁵ are as defined for general formula (I); andsalts of any of these.
 3. The compound according to claim 1 wherein R¹is ethyl.
 4. The compound according to claim 1 wherein the compound is acompound of general formula (IA) or (IB) and Y is selected from thegroup consisting of a bond, an unsubstituted C₁₋₃ alkylene group, a C₁₋₃alkylene group substituted with OH, and a C₁₋₃ alkenylene group.
 5. Thecompound according to claim 1 wherein the compound is selected from thegroup consisting of a compound of general formula (IC), a compound ofgeneral formula (ID) and a compound of general formula (IE) and Yselected from a bond and an alkylene group having 1 to 3 carbon atomsand is optionally substituted with one or two OH groups.
 6. The compoundaccording to claim 1 wherein, when present in the R⁴ moiety, each R¹⁰and R¹¹ is independently selected from: a. hydrogen and b. C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl or C₂₋₁₀ alkynyl, any of which is optionally substitutedwith one or more substituents as defined in claim 1; and c. a 6- to10-membered aryl or 5 to 10-membered heteroaryl group either of which isoptionally substituted with one or more substituents as defined in claim1; and d. a polyethylene glycol residue; or e. when R⁴ is selected fromthe group consisting of CH(OR¹⁰)(OR¹¹), CH(SR¹⁰)(SR¹¹), NR¹⁰R¹¹,BR¹⁰R¹¹, or CH[C(O)OR¹⁰]₂ or CH(BR¹⁰R¹¹)₂ an R¹⁰ and an R¹¹ group,together with the atom or atoms to which they are attached, may combineto form a 3- to 10-membered heterocyclic ring; or when R⁴ is NR¹⁰R¹¹,R¹⁰ may be selected from H and C₁₋₄ alkyl and R¹¹ may be a 5-10 memberedheteroaryl group.
 7. The compound according to claim 1 wherein: when oneor more of R², R³ and R⁵ is a protected OH group, the protected OH groupcomprises i. OC(O)R¹⁴, where R¹⁴ is a group R¹⁰; or ii. OSi(R¹⁶)₃, andwherein: when present, one or more of R¹⁰, R¹¹ and R¹⁶ is selected from:a. an alkyl, alkenyl or alkynyl group optionally substituted with one ormore substituents selected from halo, NO₂, CN, OR¹⁹, SR¹⁹, C(O)OR¹⁹,SO₂R¹⁹, SO₃R¹⁹, OSO₃R¹⁹, N(R¹⁹)₂ and a 6- to 10-membered aryl or 5 to14-membered heteroaryl group, either of which is optionally substitutedwith a group selected from C₁₋₆ alkyl, C₁₋₆ haloalkyl, halo, NO₂, CN,OR¹⁹, SO₂R¹⁹, SO₃R¹⁹ and N(R¹⁹)₂; where R¹⁹ is as defined in claim 1; orb. an aryl or heteroaryl group optionally substituted with one or moresubstituents selected from C₁₋₆ alkyl, C₁₋₆ haloalkyl, halo, NO₂, CN,OR¹⁹, SR¹⁹ and N(R¹⁹)₂; where R¹⁹ is as defined in claim
 1. 8. Thecompound according to claim 1 wherein, when present, R¹⁹ is selectedfrom H, methyl, ethyl, trifluoromethyl and phenyl optionally substitutedwith one or more substituents selected from fluoro, chloro, methyl,ethyl and trifluoromethyl.
 9. The compound according to claim 1, whereinR⁴ is selected from the group consisting of halo, CN, CH(OR¹⁰)(OR¹¹),NR¹⁰R¹¹, BR¹⁰R¹¹, —CH═CH₂, —C≡CH, CH[C(O)OR¹⁰]₂, azide, andCH(BR¹⁰R¹¹)₂; wherein R¹⁰ and R^(H) are as defined in claim
 1. 10. Thecompound according to claim 9, wherein R⁴ is selected from the groupconsisting of halo, CN, CH(OR¹⁰)(OR¹¹), NR¹⁰R¹¹, CH[C(O)OR¹⁰]₂, azideand tetrazole; wherein R¹⁰ is selected from H and C₁₋₁₀ alkyl, C₂₋₁₀alkenyl or C₂₋₁₀ alkynyl optionally substituted with one or moresubstituents selected from halo, NO₂, CN, OR¹⁹, SR¹⁹, C(O)OR¹⁹, SO₂R¹⁹,SO₃R¹⁹, OSO₃R¹⁹, N(R¹⁹)₂ and a 6- to 10-membered aryl or 5 to14-membered heteroaryl group, either of which is optionally substitutedwith one or more substituents selected from C₁₋₆ alkyl, C₁₋₆ haloalkyl,halo, NO₂, CN, OR¹⁹, SO₂R¹⁹, SO₃R¹⁹ and N(R¹⁹)₂; wherein R¹⁹ is asdefined in claim 1; or when R⁴ is CH(OR¹⁰)(OR¹¹), the OR¹⁰ and OR¹¹groups together with the carbon atom to which they are attached may forma cyclic acetal group; or when R⁴ is N¹⁰R¹¹, is selected from H and C₁₋₄alkyl and R¹¹ is a 5-10 membered heteroaryl group.
 11. The compoundaccording to claim 10, wherein R⁴ is selected from the group consistingof: Cl, Br, CN, CH(OR¹⁰)₂, 1,3-dioxane, 1,3-dioxolane, CH[C(O)OR¹⁰]₂ andtetrazole; where R¹⁰ is selected from methyl, ethyl, n-propyl,isopropyl, n-butyl, s-butyl, iso-butyl and t-butyl.
 12. The compoundaccording to claim 10 wherein R⁴ is azide.
 13. The compound according toclaim 9 wherein R⁴ is selected from —NH— tetrazole, —C(O)NHSO₂R³⁰ and—NHC(O)NHSO₂R³⁰; wherein R³⁰ is as defined in claim
 9. 14. The compoundaccording to claim 1 which is selected from the group consisting of:(6α, 7α, 20S)-20-(1-bromomethyl)-6,7-epoxy-pregn-4-en-3-one; (6α, 7α,20S)-6,7-epoxy-20-(ethylenedioxymethyl)-pregn-4-en-3-one; (6α, 7α,20S)-6,7-epoxy-20-azidomethyl-pregna-4-en-3-one; (6α,7α)-6,7-epoxy-3-oxo-4-cholen-23-carboxy-24-oic acid dimethyl ester; (6α,7α)-6-ethyl-7-hydroxy-3-oxo-4-cholen-23-carboxy-24-oic acid dimethylester; (5β, 6β, 7α)-6-ethyl-7-hydroxy-3-oxo-cholan-23-carboxy-24-oicacid dimethyl ester; (5β, 6β)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oicacid dimethyl ester; (5β, 6α)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oicacid dimethyl ester; (5β, 6α)-6-ethyl-3,7-dioxo-cholan-23-carboxy-24-oicacid; (6α, 7α)-6,7-epoxy-3-oxo-4-choleno-24-nitrile; (6β,7α)-6-ethyl-7-hydroxy-3-oxo-4-choleno-24-nitrile; (5β, 6β,7α)-6-ethyl-7-hydroxy-3-oxo-cholano-24-nitrile; (5β,6β)-3,7-dioxo-6-ethyl-cholano-24-nitrile; (3α, 5β,6β)-6-ethyl-3-hydroxy-7-oxo-cholano-24-nitrile; (6α, 7α,20R)-20-(1-cyanomethyl)-6,7-epoxy-pregn-4-en-3-one; (6β, 7α,20R)-cyanomethyl-6-ethyl-7-hydroxy-4-pregnen-3-one; (5β, 6β, 7α,20R)-cyanomethyl-6-ethyl-7-hydroxy-pregna-3-one; (5β, 6β,20R)-cyanomethyl-6-ethyl-7-oxo-pregna-3-one; (6β, 7α,20S)-20-(ethylenedioxymethyl)-6-ethyl-7-hydroxy-pregna-4-en-3-one; (5β,6β, 7α, 20S)-20-(ethylenedioxymethyl)-6-ethyl-7-hydroxy-pregna-3-one;(5β, 6β, 20S)-20-(ethylenedioxymethyl)-6-ethyl-pregna-3,7-dione; (5β,6α, 20S)-20-(ethylenedioxymethyl)-6-ethyl-pregna-3,7-dione; and saltsthereof.
 15. The compound according to claim 1, wherein the carboxylicacid mimetic group is selected from tetrazole, —C(O)NHSO₂R³⁰ and—NHC(O)NHSO₂R³⁰; wherein R³⁰ is H, C₁₋₆ alkyl or aryl.